Live attenuated virus vaccines for la crosse virus and other bunyaviridae

ABSTRACT

The invention relates to vaccine compositions including CEV serogroup immunogens, attenuated and inactivated viruses of the CEV serogroup and chimeric Bunyaviridae. Also disclosed are methods of treating or preventing CEV serogroup infection in a mammalian host, methods of producing a subunit vaccine composition or an immunogenic composition, isolated polynucleotides comprising a nucleotide sequence encoding a CEV serogroup immunogen, methods for detecting La Crosse virus (LACV) infection in a biological sample and infectious chimeric Bunyaviridae.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation of U.S. patent application Ser. No.15/850,696, filed Dec. 21, 2017, which is a Division of U.S. patentapplication Ser. No. 15/018,857, filed Feb. 8, 2016, now U.S. Pat. No.9,884,104, issued Feb. 6, 2018, which is a Division of U.S. patentapplication Ser. No. 13/633,633, filed on Oct. 2, 2012, now U.S. Pat.No. 9,255,252, issued Feb. 9, 2016, which is a Division of applicationSer. No. 12/593,818, filed Apr. 16, 2010, now U.S. Pat. No. 8,298,541,issued Oct. 30, 2012, which is the U.S. National Stage Entry ofInternational Patent Application No. PCT/US2008/056099, filed Mar. 6,2008, which claims priority to U.S. Provisional Application Nos.60/937,871, filed Jun. 29, 2007; 60/928,406, filed May 8, 2007; and60/920,961, filed Mar. 29, 2007. Each of the aforementioned patentapplications are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention pertains to immunogenic reagents derived fromviruses of the California encephalitis group of Bunyaviruses.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Jan. 4, 2019,and is ˜230 kilobytes, which is incorporated by reference herein.

DESCRIPTION OF THE RELATED ART

La Crosse virus (LACV), family Bunyaviridae, is a mosquito-bornepathogen endemic in many regions of the United States. LACV infectionresults in 70-130 clinical cases a year and is the major cause ofpediatric arboviral encephalitis in North America (Rust R S et al. 1999J Child Neurol 14:1-14; McJunkin J E et al. 2001 N Engl J Med344:801-807; Calisher C H 1994 Clin Microbiol Rev 7:89-116). LACV wasfirst identified as a human pathogen in 1960 after its isolation from a4 year-old girl from Minnesota who suffered meningoencephalitis andlater died in La Crosse, Wis. (Thompson W H and Gundersen C B 1983 ProgClin Biol Res 123:225-236; Thompson W H et al. 1965 American Journal ofEpidemiology 81:245-253). The majority of LACV infections are mild andnever reported, however serologic studies estimate annual infectionrates of 10-30/100,000 in endemic areas (McJunkin J E et al. 2001 N EnglJ Med 344:801-807; Calisher C H 1994 Clin Microbiol Rev 7:89-116;Grimstad P R et al. 1984 Am J Epidemiol 119:913-930; McJunkin J E et al.1998 Infect Dis Clin North Am 12:83-93). LACV is a member of theCalifornia encephalitis virus (CEV) serogroup of viruses in the genusOrthobunyavirus. The serogroup contains members found on five continentsthat include human pathogens such as La Crosse, Snowshoe hare, andJamestown Canyon viruses in North America; Guaroa virus in North andSouth America; Inkoo and Tahyna viruses in Europe; and Lumbo virus inAfrica (Nichol S T: “Bunyaviruses” in Fields Virology, Volume 2, Fourthedition, Edited by Knipe D M, Howley P M. Philadelphia, Pa.: LippincottWilliams & Wilkins; 2001:1603-1633). Children who recover from severe LaCrosse encephalitis may have significantly lower IQ scores than expectedand a high prevalence (60% of those tested) ofattention-deficit-hyperactivity disorder (McJunkin J E et al. 2001 NEngl J Med 344:801-807). Seizure disorders are also common in survivors(Balfour H H et al. 1973 Pediatrics 52:680-691). LACV can also causeencephalitis in immunosuppressed adults (Wurtz R and Paleologos N 2000Clin Infect Dis 31:1113-1114). Projected lifelong economic costsassociated with neurologic sequelae range from $48,775-3,090,398 percase (Utz J T et al. 2003 Am J Trop Med Hyg 69:509-518). At present, avaccine or FDA approved antiviral therapy is not available.

LACV maintains an enzootic life cycle with the hardwood forest dwelling,tree-hole mosquito, Aedes triseriatus, which lives in the eastern halfof the United States breeding in tree holes and outdoor containers(Nasci R S et al. 2000 J Med Entomol 37:559-570). Ae. triseriatusmosquitoes feed on Eastern gray squirrels (Sciurus carolinensis) andEastern chipmunks (Tamias striatus griseus) which serve as amplifyinghosts for LACV, and undergo sub-clinical infections while maintainingserum viremias high enough to infect feeding mosquitoes (Woodruff B A etal. 1992 Am J Epidemiol 136:320-327; Pantuwatana S et al. 1972 Am J TropMed Hyg 21:476-481). Interestingly, the virus can be maintained in themosquito population in the absence of vertebrate hosts by transovarial(vertical) transmission, thus allowing the virus to over-winter inmosquito eggs. Mosquito infection is lifelong and mosquitoes can becomedually infected with other bunyaviruses allowing for the development ofintra-genus reassortants (Klimas R A et al. 1981 Am J Epidemiol114:112-131; Seymour C et al. 1983 Am J Trop Med Hyg 32:1147-1153; BeatyB J et al. 1985 Science 230:548-550; Beaty B J et al. 1981 Virology111:662-665).

LACV virions are pleomorphic (90-100 nm in diameter) and have a lipidenvelope containing the heteromultimer glycoprotein (Obijeski J F et al.1976 J Virol 19:985-997). The genome consists of three single-stranded,negative-sense RNA genome segments designated small (S), medium (M), andlarge (L). Each genome segment is complexed with the nucleoprotein (N)to form three separate nucleocapsids. The termini of the 3′ and 5′non-coding regions (NCR) of each segment are complementary and highlyconserved. The Segment encodes two proteins in overlapping readingframes: the nucleoprotein (N) and a non-structural protein (NSs). In therelated Bunyamwera virus, NSs inhibits transcription via blocking hostcell RNA polymerase II, which decreases overall host cell proteinsynthesis in mammalian cells including a decrease in both the inductionof interferon and its signaling in infected cells (Bridgen A et al. 2001Proc Natl Acad Sci USA 98:664-669; Weber F et al. 2002 J Virol76:7949-7955). Recombinant LACV virions lacking the NSs gene are viable,indicating that the NSs is a nonessential accessory protein (Blakqori Gand Weber F 2005 J Virol 79:10420-10428). The M segment encodes a singlepolyprotein (M polyprotein) that is post-translationally processed intotwo glycoproteins (G_(N) and G_(C) that form a heteromultimer in thevirion and a non-structural protein (NS_(M)) of unknown function(Obijeski J F et al. 1976 J Virol 20:664-675). The L segment encodes asingle open reading frame for the RNA dependent RNA polymerase (L) (KohlA et al. 2004 J Gen Virol 85:3269-3278; Li M L et al. 1998 Embo J17:5844-5852). The L polymerase uses host-cell 5′ mRNA sequences,including the cap structures, to prime its own mRNA synthesis, a processthat also contributes to the observed shut-off of host cell proteinsynthesis following infection.

SUMMARY OF THE INVENTION

The complete genomes of three low-passage LACV isolates, namely,LACV/human/1960, LACV/human/1978, LACV/mosquito/1978, isolated over a 18year period of time, were screened to identify nucleotide sequences ofLACV that are associated with the wild type phenotype, e.g., replicationcompetent in insect and mammalian cells and able to cause encephaliticdisease in suckling and weanling mice by a peripheral and intracerebralroute of inoculation. Biologically cloned derivatives of each virus werealso sequenced. The level of neurovirulence and neuroinvasiveness foreach of the three virus isolates and their cloned derivatives wasdetermined in mice by assessing clinical disease following intracerebralor intraperitoneal administration of virus. LACV strains appear highlygenetically stable in nature, grow to high titers in monkey and mosquitocell cultures, and are highly neurovirulent and neuroinvasive for miceeven at low dosage. Since one of the long-term goals of this project wasto develop a live attenuated virus vaccine for LACV, the identificationof a nucleotide sequence of LACV that specifies a wild type phenotypewas seen as a first step in this process. Several mutations of LACV,which impact the virus were also discovered e.g., a single amino acidsubstitution in G_(N) in one of the cloned LACV strains was identifiedas a mutation that greatly decreases LACV neuroinvasiveness. Suchmutations are envisioned as providing the basis for developinglive-attenuated virus vaccines and immunogenic compositions.

Aspects of the invention relate to subunit vaccine compositionsincluding California encephalitis virus (CEV) serogroup immunogens,attenuated and inactivated CEV serogroup and chimeric Bunyaviridae. Alsodisclosed are methods of treating and/or preventing CEV serogroupinfection in a mammal, methods of inducing an immune response to animmunogenic composition described herein, methods of producing a subunitvaccine composition or an immunogenic composition, isolatedpolynucleotides comprising a nucleotide sequence encoding a CEVserogroup immunogen, methods for detecting La Crosse virus (LACV)infection in a biological sample and infectious chimeric Bunyaviridae.

Some embodiments include a live attenuated LACV vaccine or animmunogenic composition that contains a mutation at amino acid 148 ofG_(N) and, optionally, is attenuated for neuroinvasiveness in amammalian host. Preferred embodiments include a live attenuated LACVvaccine or an immunogenic composition in which the mutated amino acid atposition 148 of G_(N) is Alanine. Some aspects may concern a liveattenuated LACV vaccine or immunogenic composition in which amino acid148 of G_(N) is deleted and other aspects concern a live attenuated LACVvaccine or an immunogenic composition in which the mutated amino acid atposition 148 of G_(N) is any amino acid other than Threonine.

More embodiments involve a subunit vaccine or immunogenic compositioncomprising a CEV serogroup immunogen and a pharmaceutically acceptablevehicle, wherein the immunogen is selected from the group consisting of(a) LACV/human/1960 G_(C); (b) immunogenic fragment thereof; and (c) animmunogenic analog thereof. Some embodiments include an isolatedLACV/human/1960 virus or its LACV/human/1960-clone or sequence having atleast about 99.9% nucleotide or amino acid identity thereto. Otherembodiments include an isolated LACV/mosquito/1978 virus or itsLACV/mosquito/1978-clone or sequence having at least about 99.9%nucleotide or amino acid identity thereto. Still more embodimentsinclude an isolated LACV/human/1978 virus or its LACV/human/1978-cloneor sequence having at least about 99.99% nucleotide identity thereto.

Some aspects of the invention encompass an immunogenic compositioncomprising an attenuated LACV/human/1960 virus or itsLACV/human/1960-clone or sequence having at least about 99.9% nucleotideor amino acid identity thereto, and a pharmaceutically acceptablevehicle. Other embodiments include an immunogenic composition comprisingan attenuated LACV/mosquito/1978 virus or its LACV/mosquito/1978-cloneor sequence having at least about 99.9% nucleotide or amino acididentity thereto, and a pharmaceutically acceptable vehicle. Still moreembodiments include an immunogenic composition comprising an attenuatedLACV/human/1978 virus or its LACV/human/1978-clone or sequence having atleast about 99.99% nucleotide identity thereto, and a pharmaceuticallyacceptable vehicle. In some aspects of the invention, any one or more ofthe above immunogenic compositions has a mutation, wherein the aminoacid at position 148 in G_(N) is Alanine.

Some embodiments also include an immunogenic composition comprising anattenuated virus of the CEV serogroup and a pharmaceutically acceptablevehicle, wherein the amino acid at position 148 in G_(N) is Alanine inan exemplary manner or corresponding thereto in other viruses of the CEVserogroup. The CEV virus in the immunogenic composition above can be aLa Crosse virus. Some embodiments concern an immunogenic compositioncomprising an inactivated virus of the CEV serogroup and apharmaceutically acceptable vehicle, wherein the CEV virus is the LaCrosse virus of any of the aforementioned, preferably anLACV/mosquito/1978 immunogen comprising a mutation at amino acid atposition 148 of G_(N), an more preferably the T148A mutation.

More embodiments concern a method of treating or preventing CEVserogroup infection in a mammalian host comprising administering to saidsubject a therapeutically effective amount of any one or more of theimmunogenic compositions described above. Some embodiments encompass amethod of producing a subunit vaccine composition comprising the stepsof providing a CEV serogroup immunogen wherein the immunogen is selectedfrom the group consisting of (a) LACV/human/1960 G_(C), (b) immunogenicfragment thereof; and (c) immunogenic analog thereof; and combining saidCEV serogroup immunogen with a pharmaceutically acceptable vehicle.

Still more embodiments concern a method of producing an immunogeniccomposition comprising the steps of providing an attenuatedLACV/human/1960 virus or its LACV/human/1960-clone or sequence having atleast about 99.9% nucleotide or amino acid identity thereto; orproviding an attenuated LACV/mosquito/1978 virus or itsLACV/mosquito/1978-clone or sequence having at least about 99.9%nucleotide or amino acid identity thereto; or providing an attenuatedLACV/human/1978 virus or its LACV/human/1978-clone or sequence having atleast about 99.99% nucleotide identity thereto; and combining saidattenuated LACV with a pharmaceutically acceptable vehicle.

Aspects of the invention also include an isolated polynucleotidecomprising a nucleotide sequence encoding a CEV serogroup immunogen,wherein the immunogen is selected from the group consisting of (a)LACV/human/1960 G_(C), (b) immunogenic fragment thereof, and (c)immunogenic analog thereof.

More embodiments concern a method for detecting La Crosse virus (LACV)infection in a biological sample, the method comprising: isolatingnucleic acid from a biological sample suspected of containing LACV RNA,wherein if LACV is present, said nucleic acid comprises a targetsequence; reacting the LACV nucleic acid with a detectably labeled probesufficiently complementary to and capable of selectively hybridizingwith the target sequence, wherein said reacting is done under conditionsthat provide for the formation of a probe/target sequence complex; anddetecting the presence or absence of label as an indication of thepresence or absence of the target sequence, wherein the probe isselected from the group consisting of an oligonucleotide comprising thenucleotide substitution A503G resulting in an amino acid change atposition 148 in G_(N), and reverse complement thereof.

Still more embodiments concern a method for detecting La Crosse virus(LACV) infection in a biological sample, the method comprising:isolating nucleic acid from a biological sample suspected of containingLACV RNA, wherein if LACV is present, said nucleic acid comprises atarget sequence; amplifying the nucleic acid using at least two primerswherein each of the primers is not more than about 50 nucleotides inlength and each of the primers is sufficiently complementary to aportion of the sense and antisense strands, respectively, of LACVisolated nucleic acid, if present, to hybridize therewith, and furtherwherein at least one of the primers is capable of selectivelyhybridizing to the target sequence; and detecting the presence of theamplified nucleic acid as an indication of the presence or absence ofLACV in the sample, wherein one of the primers is selected from thegroup consisting of an oligonucleotide comprising the nucleotidesubstitution A503G resulting in an amino acid change at position 148 inG_(N), and reverse complement thereof.

Additional embodiments include a method for detecting La Crosse virus(LACV) infection in a biological sample, the method comprising:isolating nucleic acid from a biological sample suspected of containingLACV RNA, wherein if LACV is present, said nucleic acid comprises atarget sequence; amplifying the nucleic acid using at least two primerswherein each of the primers is not more than about 50 nucleotides inlength and each of the primers is sufficiently complementary to aportion of the sense and antisense strands, respectively, of LACVisolated nucleic acid, if present, to hybridize therewith; and detectingthe presence of the amplified nucleic acid using at least one detectablylabeled probe sufficiently complementary to and capable of hybridizingwith the LACV nucleic acid, if present, as an indication of the presenceor absence of LACV in the sample, wherein at least one of the primersand/or the probe is capable of selectively hybridizing to the targetsequence, wherein one of the primers is selected from the groupconsisting of an oligonucleotide comprising the nucleotide substitutionA503G resulting in an amino acid change at position 148 in G_(N), andreverse complement thereof.

Other embodiments include an infectious chimeric virus which is a memberof the Bunyaviridae family comprising a chimeric Bunyaviridae genomewhich is comprised of a La Crosse virus genome wherein one or both genesG_(C) and G_(N) of a different Bunyaviridae subgroup or strain issubstituted to replace one or both counterpart G_(C) and G_(N) genes inthe La Crosse virus genome. In some embodiments, the infectious chimericvirus above is a Jamestown Canyon Virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Alignment of 3′ non-coding region of S, M, and L genome segments(cDNA presented). S segment 3′ NCR shows highly conserved sequence withno nucleotide changes from the consensus. For each segment the consensussequence consists of three or more sequences sharing the same nucleotideat a given position (positions with no clear consensus are indicatedwith an “N”). A single nucleotide change was reported in theLACV/mosquito/1977 published sequence at position 9 of the M segment.For the 3′ NCR of the L segment, 2 changes from the consensus wereobserved in LACV/mosquito/1978 with position 31 having no clearconsensus. Underlined sequence indicates region conserved among allthree segments. Putative host-specific nucleotide sequences areindicated with an arrow (↓).

FIG. 2.1. Alignment of 5′ non-coding region of S, M, and L genomesegments (cDNA presented).

FIG. 2.2. L genome segment with nucleotides sequence 6951-6980.

Among the two human isolates only one nucleotide difference was observedin the NCR of the L segment at position 6888. For each segment, theconsensus sequence consists of three or more sequences sharing the samenucleotide at a given position (positions with no clear consensus areindicated with an “N”). Underlined sequence indicates region conservedamong all three segments. Putative host-specific nucleotide sequencesare indicated with an arrow (↓).

FIG. 3A. Growth kinetics of LACV/human/1960, LACV/human/1978, andLACV/mosquito/1978 in Vero cells or C6/36 cells infected at an MOI of0.01.

FIG. 3B. Photographs of mock or LACV/human/1960 infected Vero cellmonolayers from panel “A”. Cell rounding and detachment from the flaskcan be seen on days 2-4 post-infection in infected monolayers.

FIG. 4-1. Multiple sequence alignment of the M polyprotein of La Crossevirus isolates showing conserved regions and differences from 1-240amino acids.

FIG. 4-2. Multiple sequence alignment of the M polyprotein of La Crossevirus isolates showing conserved regions and differences from 241-480amino acids

FIG. 4-3. Multiple sequence alignment of the M polyprotein of La Crossevirus isolates showing conserved regions and differences from 481-720amino acids

FIG. 4-4. Multiple sequence alignment of the M polyprotein of La Crossevirus isolates showing conserved regions and differences from 721-960amino acids;

FIG. 4-5. Multiple sequence alignment of the M polyprotein of La Crossevirus isolates showing conserved regions and differences from 961-1200amino acids;

FIG. 4-6. Multiple sequence alignment of the M polyprotein of La Crossevirus isolates showing conserved regions and differences from 1201-1441amino acids.

“*” means that the residues or nucleotides in a given column areidentical in all sequences in the alignment. Absence of a symbol below agiven column means that a non-conservative difference is observed. “:”means that conserved substitutions are observed, and “.” means thatsemi-conserved substitutions are observed.

FIG. 5. Alignment of a portion of the M polyprotein of La Crosse virusisolates showing conservation in the sequence. Amino acid position 148is indicated by an arrow (↓).

FIG. 6. Alignment of a portion of the M polyprotein of Californiaencephalitis virus serogroup showing conservation in the sequencecompared to amino acid 148 of La Crosse virus (indicated by an arrow).LACV-CL is LACV/human/1960 with a mutation at amino acid position 148 ofthe M polyprotein, LACV is wild-type LACV/human/1960. CEV=Californiaencephalitis virus, IV=Inkoo virus, JCV=Jamestown Canyon virus,SHV=Snowshoe hare virus, TV=Tahyna virus, SDNV=Serra do Navio virus,JSV=Jerry Slough virus, SAV=San Angelo virus, LV=Lumbo virus, MV=Melaovirus, TVTV=Trivittatus virus, SRV=South River virus, and KV=Keystonevirus.

BRIEF DESCRIPTION OF THE APPENDICES

Appendix 1. Large genomic segment-nucleotide sequence alignment ofLACV/human/1978 (Hughes et al. 2002) and LACV/human/1978 (this study).

Appendix 2. Medium genomic segment-nucleotide sequence alignment ofLACV/human/1978 (Hughes et al. 2002) and LACV/human/1978 (this study).

Appendix 3. Small genomic segment-nucleotide sequence alignment ofLACV/human/1978 (Hughes et al. 2002) and LACV/human/1978 (this study).

Appendix 4. Large genomic segment-amino acid sequence alignment ofLACV/human/1978 (Hughes et al. 2002) and LACV/human/1978 (this study).

Appendix 5. Medium genomic segment-amino acid sequence alignment ofLACV/human/1978 (Hughes et al. 2002) and LACV/human/1978 (this study).

Appendix 6. Small genomic segment-Nucleoprotein (N) amino acid sequencealignment of LACV/human/1978 (Hughes et al. 2002) and LACV/human/1978(this study).

Appendix 7. Small genomic segment-Nonstructural protein (NSs) amino acidsequence alignment of LACV/human/1978 (Hughes et al.) andLACV/human/1978 (this study).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g., Singleton P andSainsbury D., Dictionary of Microbiology and Molecular Biology 3rd ed.,J. Wiley & Sons, Chichester, N.Y., 2001, and Fields Virology 5th ed.,Knipe D. M. and Howley P. M. eds, Lippincott Williams & Wilkins,Philadelphia 2007.

The transitional term “comprising” is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps.

The transitional phrase “consisting of excludes any element, step, oringredient not specified in the claim, but does not exclude additionalcomponents or steps that are unrelated to the invention such asimpurities ordinarily associated therewith.

The transitional phrase “consisting essentially of limits the scope of aclaim to the specified materials or steps and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also includepost-expression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions andsubstitutions (generally conservative in nature), to the nativesequence, so long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

A CEV serogroup polypeptide is a polypeptide, as defined above, derivedfrom a virus of the California encephalitis (CEV) serogroup of the genusBunyavirus, including, without limitation, any of the various isolatesof the California encephalitis group of viruses such as Californiaencephalitis, Guaroa, Inkoo, Jamestown Canyon, La Crosse, Snowshoe hare,and Tahyna (Lumbo) virus. The polypeptide need not be physically derivedfrom the particular isolate in question, but may be synthetically orrecombinantly produced.

Sequences for polypeptides and the nucleic acid sequences encoding for anumber of CEV serogroup are known. Representative sequences arepresented herein for LACV polypeptides. Refer to Campbell et al. 1999Virus Res 61:137-144, for a comparison of M RNA among the CEV serogroupof viruses.

The terms “analog” and “mutein” refer to biologically active derivativesof the reference molecule, that retain desired activity, such asimmunoreactivity in assays described herein, and/or the capability ofeliciting an immune response as defined below, such as the ability toelicit neutralizing antibodies. In general, the term “analog” refers tocompounds having a native polypeptide sequence and structure with one ormore amino acid additions, substitutions (generally conservative innature) and/or deletions, relative to the native molecule, so long asthe modifications do not destroy immunogenic activity and which are“substantially homologous” to the reference molecule as deemed below. Anumber of conserved and variable regions are known between the variousisolates and, in general, the amino acid sequences of epitopes derivedfrom these regions will have a high degree of sequence homology, e.g.,amino acid sequence homology of more than 50%, generally more than60%-70%, when the two sequences are aligned. The term “mutein” refers topeptides having one or more peptide mimics (“peptoids”). Preferably, theanalog or mutein has at least the same immunoreactivity as the nativemolecule. Methods for making M polypeptide analogs and muteins are knownin the art and are described further below.

Particularly preferred analogs include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine, threonine, and tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25, 50or 75 conservative or non-conservative amino acid substitutions, or anyinteger between 5-75, so long as the desired function of the moleculeremains intact. One of skill in the art can readily determine regions ofthe molecule of interest that can tolerate change by reference toKyte-Doolittle plots, well known in the art.

By “fragment” is intended a polypeptide consisting of only a part of theintact full-length polypeptide sequence and structure. The fragment caninclude a C-terminal deletion, an N-terminal deletion, and/or aninternal deletion of the native polypeptide.

By a “M polypeptide” is meant a polypeptide, as defined above, encodedby the M region of the CEV serogroup in question. As explained above,the M region encodes the G_(C) and G_(N) polypeptides (also referred toformerly as G1 and G2), as well as the NS_(M) polypeptide. Thenucleotide and corresponding amino acid sequences for various CEVserogroup M regions are known. Refer to Campbell et al., supra.

As explained above, G_(C) and/or G_(N) polypeptides for use with thepresent invention include the full-length or substantially full-lengthproteins, as well as fragments, fusions of G_(C) and G_(N) polypeptides,or mutants of the proteins, which include one or more epitopes such thatimmunological activity is retained.

An “antigen” refers to a molecule, such as a polypeptide as deemedabove, containing one or more epitopes (either linear, conformational orboth) that will stimulate a host's immune system to make a humoraland/or cellular antigen-specific response. The term is usedinterchangeably with the term “immunogen.” Normally, a B-cell epitopewill include at least about 5 amino acids but can be as small as 3-4amino acids. A T-cell epitope, such as a CTL epitope, will include atleast about 7-9 amino acids, and a helper T-cell epitope at least about12-20 amino acids. Normally, an epitope will include between about 7 and15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term “antigen”denotes both subunit antigens, (i.e., antigens which are separate anddiscrete from a whole organism with which the antigen is associated innature), as well as, killed, attenuated or inactivated viruses.Antibodies such as anti-idiotype antibodies, or fragments thereof, andsynthetic peptide mimotopes, which can mimic an antigen or antigenicdeterminant, are also captured under the definition of antigen as usedherein. Similarly, an oligonucleotide or polynucleotide that expressesan antigen or antigenic determinant in vivo, such as in nucleic acidimmunization applications, is also included in the definition of antigenherein.

For purposes of the present invention, immunogens can be derived fromany of several known viruses of the CEV serogroup, as described above,for example LACV. By “immunogenic fragment” is meant a fragment of a CEVserogroup polypeptide that includes one or more epitopes and thuselicits one or more of the immunological responses described herein. An“immunogenic fragment” of a particular CEV serogroup protein willgenerally include at least about 5-10 contiguous amino acid residues ofthe full-length molecule, preferably at least about 15-25 contiguousamino acid residues of the full-length molecule, and most preferably atleast about 20-50 or more contiguous amino acid residues of thefull-length molecule, that define an epitope, or any integer between 5amino acids and the full-length sequence, provided that the fragment inquestion retains the ability to elicit an immunological response asdefined herein.

The term “epitope” as used herein refers to a sequence of at least about3 to 5, preferably about 5 to 10 or 15, and not more than about 500amino acids (or any integer therebetween), which define a sequence thatby itself or as part of a larger sequence, elicits an immunologicalresponse in the subject to which it is administered. Often, an epitopewill bind to an antibody generated in response to such sequence. Thereis no critical upper limit to the length of the epitope, which maycomprise nearly the full-length of the protein sequence, or even afusion protein comprising two or more epitopes from the CEV serogroupmolecule in question. An epitope for use in the subject invention is notlimited to a polypeptide having the exact sequence of the portion of theparent protein from which it is derived. Indeed, viral genomes are in astate of constant flux and contain several variable domains whichexhibit relatively high degrees of variability between isolates. Thusthe term “epitope” encompasses sequences identical to the nativesequence, as well as modifications to the native sequence, such asdeletions, additions and substitutions (generally conservative innature).

Regions of a given polypeptide that include an epitope can be identifiedusing any number of epitope mapping techniques, well known in the art.For example, linear epitopes may be determined by e.g., concurrentlysynthesizing large numbers of peptides on solid supports, the peptidescorresponding to portions of the protein molecule, and reacting thepeptides with antibodies while the peptides are still attached to thesupports. Similarly, conformational epitopes are readily identified bydetermining spatial conformation of amino acids such as by, e.g., x-raycrystallography and 2-dimensional nuclear magnetic resonance. Antigenicregions of proteins can also be identified using standard antigenicityand hydropathy plots. One can use computer programs that employ theKyte-Doolittle technique for hydropathy plots.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectercells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular immunogen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. Methodsof measuring cell-mediated immune response include measurement ofintracellular cytokines or cytokine secretion by T-cell populations, orby measurement of epitope specific T-cells.

Thus, an immunological response as used herein may be one thatstimulates the production of antibodies (e.g., neutralizing antibodiesthat block viruses of the CEV serogroup from entering cells and/orreplicating by binding to the pathogens, typically protecting cells frominfection and destruction). The antigen of interest may also elicitproduction of CTLs. Hence, an immunological response may include one ormore of the following effects: the production of antibodies by B-cells;and/or the activation of suppressor T-cells and/or δγ T-cells directedspecifically to an antigen or antigens present in the composition orvaccine of interest. These responses may serve to neutralizeinfectivity, and/or mediate antibody-complement, or antibody dependentcell cytotoxicity (ADCC) to provide protection to an immunized host.Such responses can be determined using standard immunoassays andneutralization assays, well known in the art. Moreover, theimmunogenicity of the various polypeptides and polynucleotides describedherein can be tested in appropriate animal models. Acceptable animalmodels for studying viruses of the CEV serogroup are known in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest. The immunogeniccomposition can be introduced directly into a recipient subject, such asby injection, inhalation, oral, intranasal and mucosal (e.g.,intra-rectally or intra-vaginally) administration. An “immunogeniccomposition” also denotes a composition for use in diagnostic assays,described further below.

By “subunit vaccine” is meant a vaccine composition that includes one ormore selected antigens but not all antigens, derived from or homologousto, an antigen from the CEV serogroup, such as LACV. Such a compositionis substantially free of intact virus or viral particles. Thus, a“subunit vaccine” can be prepared from at least partially purified(preferably substantially purified) immunogenic polypeptides from thepathogen, or analogs thereof. The method of obtaining an antigenincluded in the subunit vaccine can thus include standard purificationtechniques, recombinant production, or synthetic production.

“Substantially purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

By “equivalent antigenic determinant” is meant an antigenic determinantfrom different isolates or strains of the CEV serogroup which antigenicdeterminants are not necessarily identical due to sequence variation,but which occur in equivalent positions in the CEV serogroup sequence inquestion. In general the amino acid sequences of equivalent antigenicdeterminants will have a high degree of sequence homology, e.g., aminoacid sequence homology of more than 30%, usually more than 40%, such asmore than 60%, and even more than 80-90% homology, when the twosequences are aligned.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two nucleic acid, or two polypeptide sequencesare “substantially homologous” to each other when the sequences exhibitat least about 50%, preferably at least about 75%, more preferably atleast about 80%-85%, preferably at least about 90%, and most preferablyat least about 99.9%-99.99% sequence identity over a defined length ofthe molecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified sequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure, M. O. Dayhoff ed., vol. 5(Suppl. 3):353-358, National Biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and Waterman1981 Advances in Appl Math 2:482-489, for peptide analysis. Programs fordetermining nucleotide sequence identity are available in the WisconsinSequence Analysis Package, Version 8 (available from Genetics ComputerGroup, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs,which also rely on the Smith and Waterman algorithm. These programs arereadily utilized with the default parameters recommended by themanufacturer and described in the Wisconsin Sequence Analysis Packagereferred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used herein to include a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. It also includes modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing non-nucleotidicbackbones, for example, polyamide (e.g., peptide nucleic acids (PNAs))and polymorpholino (commercially available from the AVI Biopharma,Corvallis, Oreg., as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration, which allows for base pairingand base stacking, such as is found in DNA and RNA. There is no intendeddistinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms will be used interchangeably. Thus, these terms include, forexample, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, DNA:RNAhybrids, and hybrids between PNAs and DNA or RNA, and also include knowntypes of modifications, for example, labels, which are known in the art,methylation, “caps,” substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide or oligonucleotide. In particular, DNA isdeoxyribonucleic acid.

A polynucleotide “derived from” a designated sequence refers to apolynucleotide sequence which comprises a contiguous sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10-12 nucleotides, and evenmore preferably at least about 15-20 nucleotides corresponding, i.e.,identical or complementary to, a region of the designated nucleotidesequence. The derived polynucleotide will not necessarily be derivedphysically from the nucleotide sequence of interest, but may begenerated in any manner, including, but not limited to, chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. As such, it mayrepresent either a sense or an antisense orientation of the originalpolynucleotide.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed andtranslated into a polypeptide in vitro or in vivo when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Atranscription termination sequence may be located 3′ to the codingsequence.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their desiredfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper transcription factors, etc., are present. The promoter need notbe contiguous with the coding sequence, so long as it functions todirect the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between thepromoter sequence and the coding sequence, as can transcribed introns,and the promoter sequence can still be considered “operably linked” tothe coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

A “control element” refers to a polynucleotide sequence which aids inthe expression of a coding sequence to which it is linked. The termincludes promoters, transcription termination sequences, upstreamregulatory domains, polyadenylation signals, untranslated regions,including 5′-UTRs and 3¹-UTRs and when appropriate, leader sequences andenhancers, which collectively provide for the transcription andtranslation of a coding sequence in a host cell.

A “promoter” as used herein is a regulatory region capable of bindingRNA polymerase in a host cell and initiating transcription of adownstream (3′ direction) coding sequence operably linked thereto. Forpurposes of the present invention, a promoter sequence includes theminimum number of bases or elements necessary to initiate transcriptionof a gene of interest at levels detectable above background. Within thepromoter sequence is a transcription initiation site, as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. Eukaryotic promoters will often, but not always, contain“TATA” boxes and “CAT” boxes.

A control sequence “directs the transcription” of a coding sequence in acell when RNA polymerase will bind the promoter sequence and transcribethe coding sequence into mRNA, which is then translated into thepolypeptide encoded by the coding sequence.

“Expression cassette” or “expression construct” refers to an assemblywhich is capable of directing the expression of the sequence(s) orgene(s) of interest. The expression cassette includes control elements,as described above, such as a promoter which is operably linked to (soas to direct transcription of) the sequence(s) or gene(s) of interest,and often includes a polyadenylation sequence as well. Within certainembodiments of the invention, the expression cassette described hereinmay be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude, one or more selectable markers, a signal which allows theplasmid construct to exist as single-stranded DNA (e.g., a M 1 3 originof replication), at least one multiple cloning site, and a “mammalian”origin of replication (e.g., a SV40 or adenovirus origin ofreplication).

“Transformation” as used herein, refers to the insertion of an exogenouspolynucleotide into a host cell, irrespective of the method used forinsertion: for example, transformation by direct uptake, transfection,infection, and the like. For particular methods of transfection, seefurther below. The exogenous polynucleotide may be maintained as anonintegrated vector, for example, an episome, or alternatively, may beintegrated into the host genome.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected immunogens into a host cell,for the in vivo expression of the immunogen. The nucleic acid moleculecan be introduced directly into a recipient subject, such as byinjection, electroporation, inhalation, oral, intranasal and mucosaladministration, or the like, or can be introduced ex vivo, into cellswhich have been removed from the host. In the latter case, thetransformed cells are reintroduced into the subject where an immuneresponse can be mounted against the immunogen encoded by the nucleicacid molecule.

An “antibody” intends a molecule that, through chemical or physicalmeans, specifically binds to a polypeptide of interest. Thus, ananti-LACV O_(N) antibody is a molecule that specifically binds to anepitope of a LACV O_(N) protein. The term “antibody” as used hereinincludes antibodies obtained from both polyclonal and monoclonalpreparations, as well as, the following: hybrid (chimeric) antibodymolecules; F(ab′)₂ and F(ab) fragments; Fv molecules (non-covalentheterodimers; single-chain Fv molecules (sFv); dimeric and trimericantibody fragment constructs; minibodies; humanized antibody molecules;and, any functional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule.

As used herein, a “solid support” refers to a solid surface such as amagnetic bead, latex bead, microtiter plate well, glass plate, nylon,agarose, acrylamide, and the like. “Immunologically reactive” means thatthe antigen in question will react specifically with anti-CEV serogroupantibodies present in a biological sample from a CEV-infectedindividual.

“Immune complex” intends the combination formed when an antibody bindsto an epitope on an antigen.

As used herein, the term “target nucleic acid region” or “target nucleicacid” denotes a nucleic acid molecule with a “target sequence” to beamplified. The target nucleic acid may be either single-stranded ordouble-stranded and may include other sequences besides the targetsequence, which may not be amplified. The term “target sequence” refersto the particular nucleotide sequence of the target nucleic acid whichis to be amplified. The target sequence may include a probe-hybridizingregion contained within the target molecule with which a probe will forma stable hybrid under desired conditions. The “target sequence” may alsoinclude the complexing sequences to which the oligonucleotide primerscomplex and extended using the target sequence as a template. Where thetarget nucleic acid is originally single-stranded, the term “targetsequence” also refers to the sequence complementary to the “targetsequence” as present in the target nucleic acid. If the “target nucleicacid” is originally double-stranded, the term “target sequence” refersto both the plus (+) and minus (−) strands.

The term “primer” or “oligonucleotide primer” as used herein, refers toan oligonucleotide which acts to initiate synthesis of a complementarynucleic acid strand when placed under conditions in which synthesis of aprimer extension product is induced, i.e., in the presence ofnucleotides and a polymerization-inducing agent such as a DNA or RNApolymerase and at suitable temperature, pH, metal concentration, andsalt concentration. The primer is preferably single-stranded for maximumefficiency in amplification, but may alternatively be double-stranded.If double-stranded, the primer can first be treated to separate itsstrands before being used to prepare extension products. Thisdenaturation step is typically effected by heat, but may alternativelybe carried out using alkali, followed by neutralization. Thus, a“primer” is complementary to a template, and complexes by hydrogenbonding or hybridization with the template to give a primer/templatecomplex for initiation of synthesis by a polymerase, which is extendedby the addition of covalently bonded bases linked at its 3′ endcomplementary to the template in the process of DNA or RNA synthesis.

As used herein, the term “probe” or “oligonucleotide probe” refers to astructure comprised of a polynucleotide, as defined above, that containsa nucleic acid sequence complementary to a nucleic acid sequence presentin the target nucleic acid analyte. The polynucleotide regions of probesmay be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.Probes may be labeled in order to detect the target sequence. Such alabel may be present at the 5′ end, at the 3′ end, at both the 5′ and 3′ends, and/or internally. For example, when an “oligonucleotide probe” isto be used in a 5′ nuclease assay, such as the TaqMan™ technique, theprobe will contain at least one fluorescer and at least one quencherwhich is digested by the 5′ endonuclease activity of a polymerase usedin the reaction in order to detect any amplified target oligonucleotidesequences. In this context, the oligonucleotide probe will have asufficient number of phosphodiester linkages adjacent to its 5′ end sothat the 5′ to 3′ nuclease activity employed can efficiently degrade thebound probe to separate the fluorescers and quenchers.

As used herein, the term “capture oligonucleotide” refers to anoligonucleotide; that contains a nucleic acid sequence complementary toa nucleic acid sequence present in the target nucleic acid analyte suchthat the capture oligonucleotide can “capture” the target nucleic acid.One or more capture oligonucleotides can be used in order to capture thetarget analyte. The polynucleotide regions of a capture oligonucleotidemay be composed of DNA, and/or RNA, and/or synthetic nucleotide analogs.By “capture” is meant that the analyte can be separated from othercomponents of the sample by virtue of the binding of the capturemolecule to the analyte. Typically, the capture molecule is associatedwith a solid support, either directly or indirectly.

It will be appreciated that the hybridizing sequences need not haveperfect complementarily to provide stable hybrids. In many situations,stable hybrids will form where fewer than about 10% of the bases aremismatches, ignoring loops of four or more nucleotides. Accordingly, asused herein the term “complementary” refers to an oligonucleotide thatforms a stable duplex with its “complement” under assay conditions,generally where there is about 90% or greater homology.

The terms “hybridize” and “hybridization” refer to the formation ofcomplexes between nucleotide sequences which are sufficientlycomplementary to form complexes via Watson-Crick base pairing. Where aprimer “hybridizes” with target (template), such complexes (or hybrids)are sufficiently stable to serve the priming function required by, e.g.,the DNA polymerase to initiate DNA synthesis.

As used herein, the term “binding pair” refers to first and secondmolecules that specifically bind to each other, such as complementarypolynucleotide pairs capable of forming nucleic acid duplexes. “Specificbinding” of the first member of the binding pair to the second member ofthe binding pair in a sample is evidenced by; the binding of the firstmember to the second member, or vice versa, with greater affinity andspecificity than to other components in the sample. The binding betweenthe members of the binding pair is typically noncovalent. Unless thecontext clearly indicates otherwise, the terms “affinity molecule” and“target analyte” are used herein to refer to first and second members ofa binding pair, respectively.

The terms “specific-binding molecule” and “affinity molecule” are usedinterchangeably herein and refer to a molecule that will selectivelybind, through chemical or physical means to a detectable substancepresent in a sample. By “selectively bind” is meant that the moleculebinds preferentially to the target of interest or binds with greateraffinity to the target than to other molecules. For example, a DNAmolecule will bind to a substantially complementary sequence and not tounrelated sequences. An oligonucleotide that “specifically binds” to aLACV sequence denotes an oligonucleotide, e.g., a primer, probe or acapture oligonucleotide, that binds to a LACV sequence but does not bindto a sequence from other viruses of the CEV serogroup.

The “melting temperature” or “Tm” of double-stranded DNA is defined asthe temperature at which half of the helical structure of DNA is lostdue to heating or other dissociation of the hydrogen bonding betweenbase pairs, for example, by acid or alkali treatment, or the like. TheTm of a DNA molecule depends on its length and on its base composition.DNA molecules rich in G_(C) base pairs have a higher Tm than thosehaving an abundance of AT base pairs. Separated complementary strands ofDNA spontaneously reassociate or anneal to form duplex DNA when thetemperature is lowered below the Tm. The highest rate of nucleic acidhybridization occurs approximately 25° C. below the Tm. The Tm may beestimated using the following relationship: Tm=69.3+0.41 (GC) %.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, including but not limited to, forexample, blood, plasma, serum, fecal matter, urine, bone marrow, bile,spinal fluid, lymph fluid, samples of the skin, external secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, organs, biopsies and also samples of in vitrocell culture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,semiconductor nanoparticles, dyes, metal ions, ligands (e.g., biotin,streptavidin or haptens) and the like. The term “fluoresces” refers to asubstance or a portion thereof which is capable of exhibitingfluorescence in the detectable range. Particular examples of labelswhich may be used under the invention include, but are not limited to,horse radish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl,umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol,NADPH and β-galactosidase.

The terms “effective amount” or “pharmaceutically effective amount” ofan immunogenic composition, as provided herein, refer to a nontoxic butsufficient amount of the composition to provide the desired response,such as an immunological response, and optionally, a correspondingtherapeutic effect. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the condition being treated, and the particularmacromolecule of interest, mode of administration, and the like. Anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

The term “treatment” as used herein refers to either (1) the preventionof infection or reinfection (prophylaxis), or (2) the reduction orelimination of symptoms of the disease of interest (therapy).

By “mammalian host” is meant any mammal susceptible to infection withthe particular member of the CEV serogroup in question. Such mammalsinclude, without limitation, humans and other primates, includingnon-human primates such as chimpanzees and other apes and monkeyspecies; rodents such as chipmunks, squirrels and laboratory animalsincluding mice, rats and guinea pigs; rabbits, hares (such as thesnowshoe hare); and domestic animals such as dogs and cats. The termdoes not denote a particular age. Thus, both adult and newborn subjectsare intended to be covered. The invention described herein is intendedfor use in any of the above mammalian species, since the immune systemsof all of these mammals operate similarly.

Bunyaviridae Viruses

Many viruses in the Bunyaviridae family are significant human,veterinary, and plant pathogens. The family is divided into five genera:Bunyavirus, Phlebovirus, Nairovirus, Hantavirus, and Tospovirus. All ofthe viruses, with the exception of the hantaviruses, are vector-borne.All infect vertebrates with the exception of Tospoviruses, which areplant viruses. The genera are further divided into serogroups based onantigenic relationships. La Crosse Virus (LACV) is a member of the genusBunyavirus, California encephalitis virus (CEV) serogroup, and is amajor cause of encephalitis and aseptic meningitis in children in theUnited States. The CEV serogroup includes other viruses that are knownto cause human illness, such as Jamestown Canyon, Californiaencephalitis, snowshoe hare (SSH), Tahyna, Inkoo, and Guaroa virus.Notable members of the Bunyaviridae family are listed in Table A.

TABLE A Notable Bunyaviridae family members Genus Main Groups NotableVirus Members Bunyavirus Anopheles A group Tacaiuma Virgin RiverAnopheles B group Anopheles B Bakau group Bakau Bunyamwera groupBunyamwera Cache Valley Fort Sherman Germiston Ilesha Kairi Main drainShokwe Wyeomyia Xingu Bwamba group Bwamba Pongola Group C Apeu CarapaniItaqui Madrid Marituba Murutucu Nepuyo Oriboca Ossa Restan Californiaencephalitis group California encephalitis Guaroa Inkoo Jamestown CanyonLa Crosse Snowshoe hare Tahyna (Lumbo) Capim group Capim Gamboa groupGamboa Guama group Catu Guama Koongol group Koongol Minatitlan groupMinatitlan Nyando group Nyando Olifantsvlei group Olifantsvlei Patoisgroup Patois Estero Real Simbu group Akabane Ingwavuma Oropouche Tetegroup Bahig Weldona Turlock Turlock Phlebovirus Sandfly fever groupCandiru Punta Toro Rift Valley fever Sandfly fever Naples ToscanaSandfly fever Sicilian Chagres Uukuniemi group Uukuniemi NairovirusCrimean-Congo HF group Crimean-Congo hemorrhagic fever Hazara NairobiSheep Disease Group Nairobi sheep disease Dugbe Dera Ghazi Khan groupDera Ghazi Khan Hughes group Hughes Qalyub group Qalyub Sakhalin groupSakhalin Thiafora group Thiafora Hantavirus Hantaan group HantaanDobrava Seoul Puumala group Puumala Sin Nombre Group Sin Nombre BayouBlack Creek Canal Andes Laguna Negra Juquitiba Araraquara Castelo dosSonhos Choclo Thottapalayam group Thottapalayam Tospovirus Tomatospotted wilt Impatiens necrotic spot Groundnut bud necrosis

The Bunyaviruses have tripartite, negative-sense RNA genomes. Consensusnucleotide sequences are present in the 3′ termini of all three RNAsegments and cause the segment ends to bind to complementary sequenceson 5′ ends to form panhandle structures. The consensus sequences mayserve as transcriptase recognition structures.

The genomic coding strategies of LAC virus are known: the large (L) RNAsegment (6980 nucleotides (nt)) codes for the polymerase; the medium (M)RNA segment (4526 nt) codes for a polyprotein that ispost-translationally processed, yielding the G_(C) and G_(N)glycoproteins (also referred to formerly as G1 and G2) and anonstructural protein NS_(M); and the small (S) RNA segment (984 nt)codes for the nucleocapsid (N) protein and a nonstructural NSs inoverlapping reading frames.

The pleomorphic virion is approximately 90-100 nm in diameter. Thevirion consists of the three genome segments encapsidated with the Nprotein to form helical structures, a few nucleocapsid-associatedpolymerase molecules needed to transcribe the negative-sense viralgenome, and a surrounding host-derived lipid envelope containingvirus-encoded glycoprotein spikes.

Aspects of the present invention are based on the discovery of reagentsand methods for preventing, treating and diagnosing infection caused bythe CEV serogroup of viruses, such as LACV infection. The methods useattenuated or inactivated viruses, or subunit compositions, to treat orprevent infection. Moreover, polypeptides and polynucleotides derivedfrom the CEV serogroup can be used in diagnostic assays to identifyinfected subjects.

A more detailed discussion is provided below regarding viruses of theCEV serogroup, various CEV polypeptide and polynucleotide immunogens foruse in the subject compositions and methods, as well as, production ofthe proteins, antibodies thereto and methods of using the proteins andantibodies.

CEV Subgroup Polypeptides and Polynucleotides

As explained above, the CEV serogroup family of viruses belongs to theBunyavirus genus and are enveloped, minus-sense RNA viruses. The RNA ofthe viral genome is tripartite, consisting of three fragments generallydesignated as S, M and L for small, medium and large genome fragments,respectively. The M segment encodes two envelope glycoproteins, termedG_(C) and G_(N), and a nonstructural protein (NS_(M)), in a single openreading frame. The S segment encodes a nucleocapsid protein, termed Nand an additional nonstructural protein termed NSs, in overlappingreading frames. The L segment of the genome encodes an RNA-dependent RNApolymerase.

Several distinct viruses of the CEV serogroup are found in associationwith specific mammalian hosts worldwide. Polypeptides andpolynucleotides derived from any of the various isolates of the CEVserogroup will find use herein, including without limitation, any of theCalifornia encephalitis group of viruses such as Californiaencephalitis, Guaroa, Inkoo, Jamestown Canyon, La Crosse, Snowshoe hare,and Tahyna (Lumbo) virus.

Thus, immunogens for use in subunit vaccines and diagnostics includethose derived from one or more of the above regions from any CEVserogroup strain or isolate. Either the full-length proteins, fragmentsthereof containing epitopes of the full-length proteins, as well asfusions of the various regions or fragments thereof, will find use inthe subject compositions and methods. Thus, for example, immunogens foruse in such compositions can be derived from the G_(C) and/or G_(N)envelope regions of any of these CEV serogroup isolates. Immunogenicfragments of the envelope proteins, which comprise epitopes may be usedin the subject compositions and methods. For example, fragments of theG_(C) and/or G_(N) polypeptide can comprise from about 5 contiguousamino acids to nearly the full-length of the molecule (e.g., at least orequal to 6, 10, 25, 50, 75, 100, 200, 250, 300, 350, 400, 450 or morecontiguous amino acids of a G_(C) and/or G_(N) polypeptide, or anyinteger between the stated numbers). Additionally, the entire M region,including G_(C), G_(N) and NSm, as well as, complexes of the G_(C) andG_(N) polypeptides, with or without NSm, or epitopes from the G_(C)polypeptide fused to epitopes of the G_(N) polypeptide with or withoutNSm, can be used in the subject compositions and methods.

Moreover, the G_(C) and/or G_(N) polypeptides for use herein may lackall or a portion of the transmembrane binding domain and/or thecytoplasmic tail found in the C-terminus of the envelope. Thus, aspectsof the present invention contemplate the use of envelope polypeptideswhich retain the transmembrane binding domain and cytoplasmic tail, aswell as polypeptides, which lack all or a portion of the transmembranebinding domain and/or the cytoplasmic tail. The location of such domainscan be readily determined using computer programs and algorithms wellknown in the art, such as the Kyte-Doolittle technique.

Polynucleotides and polypeptides for use with the some embodiments canbe obtained using standard techniques. For example, polynucleotidesencoding the various immunogenic polypeptides can be isolated from agenomic library derived from nucleic acid sequences present in, forexample, the plasma, serum, or tissue homogenate of a CEVserogroup-infected individual. Additionally, the nucleic acid can beobtained directly from the virus in question.

Alternatively, viruses of the CEV serogroup can be isolated frominfected mosquitos. Once obtained, the virus can be propagated usingknown techniques, such as described in Pekosz et al. 1995 J Virol69:3475-3481. Generally, viruses of the CEV serogroup are grown in Veroor BHK cell lines. An amplification method such as PCR can be used toamplify polynucleotides from either CEV serogroup genomic RNA or cDNAencoding therefor. Alternatively, polynucleotides can be synthesized inthe laboratory, for example, using an automatic synthesizer.

Polynucleotides can comprise coding sequences for the variouspolypeptides which occur naturally or can include artificial sequenceswhich do not occur in nature. These polynucleotides can be ligated toform a coding sequence for a fusion protein, if desired, using standardmolecular biology techniques.

Once coding sequences have been prepared or isolated, such sequences canbe cloned into any suitable vector or replicon. Numerous cloning vectorsare known to those of skill in the art, and the selection of anappropriate cloning vector is a matter of choice. Suitable vectorsinclude, but are not limited to, plasmids, phages, transposons, cosmids,chromosomes or viruses which are capable of replication when associatedwith the proper control elements. The coding sequence is then placedunder the control of suitable control elements, depending on the systemto be used for expression. Thus, the coding sequence can be placed underthe control of a promoter, ribosome binding site (for bacterialexpression) and, optionally, an operator, so that the DNA sequence ofinterest is transcribed into RNA by a suitable transformant. The codingsequence may or may not contain a signal peptide or leader sequencewhich can later be removed by the host in post-translational processing.

If present, the signal sequence can be the native leader found inassociation with the CEV serogroup polypeptide of interest. For example,if the CEV serogroup polypeptide being expressed is the CEV serogroupG_(N) polypeptide, all or a portion of the native G_(N) leader sequencecan be included.

Alternatively, a heterologous signal sequence can be present which canincrease the efficiency of secretion. A number of representative leadersequences are known in the art and include, without limitation, theyeast a-factor leader, the TPA signal peptide, the Ig signal peptide,and the like. Sequences for these and other leader sequences are wellknown in the art.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of the sequencesrelative to the growth of the host cell. Regulatory sequences are knownto those of skill in the art, and examples include those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of a regulatory compound.Other types of regulatory elements may also be present in the vector.For example, enhancer elements may be used herein to increase expressionlevels of the constructs. Examples include the SV40 early gene enhancer,the enhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus and elements derived from human CMV, such as elementsincluded in the CMV intron A sequence. The expression cassette mayfurther include an origin of replication for autonomous replication in asuitable host cell, one or more selectable markers, one or morerestriction sites, a potential for high copy number and a strongpromoter.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the “control” of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the sequences encodingthe molecule of interest may be desirable to achieve this end. Forexample, in some cases it may be necessary to modify the sequence sothat it can be attached to the control sequences in the appropriateorientation; i.e., to maintain the reading frame. The control sequencesand other regulatory sequences may be ligated to the coding sequenceprior to insertion into a vector. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

As explained above, it may also be desirable to produce mutants oranalogs of the polypeptide of interest. Mutants or analogs of CEVserogroup polynucleotides and polypeptides for use in the subjectcompositions may be prepared by the deletion of a portion of thesequence encoding the molecule of interest, by insertion of a sequence,and/or by substitution of one or more nucleotides within the sequence.Techniques for modifying nucleotide sequences, such as site-directedmutagenesis, and the like, are well known to those skilled in the art.It is also to be understood that any of the mutations described hereincan be introduced into virus that are related to the LACV virusesdescribed herein at conserved positions (e.g., position 148) so as tocreate attenuated virus, vaccines, and immunogenic compositions. It isalso contemplated that these mutations can be introduced within 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 amino acid positions before or after theidentified sites of mutation described herein (e.g., 148) and obtain anattenuated virus, vaccine, or immunogenic composition. In order tofacilitate recombinant expression, the molecule of interest can beexpressed as a fusion protein.

The molecules can be expressed in a wide variety of systems, includinginsect, mammalian, bacterial, viral and yeast expression systems, allwell known in the art. For example, insect cell expression systems, suchas baculovirus systems, are known to those of skill in the art anddescribed in, e.g., Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987). Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit).Similarly, bacterial and mammalian cell expression systems are wellknown in the art and described in, e.g., Sambrook, J., Russell, D. W.(2001) Molecular Cloning: A Laboratory Manual, the third edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Yeastexpression systems are also known in the art and described in, e.g.,Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths,London.

A number of appropriate host cells for use with the above systems arealso known. For example, mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, Vero cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human embryonic kidney cells, humanhepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney(“MDBK”) cells, as well as others. Similarly, bacterial hosts such as E.coli, Bacillus subtilis, and Streptococcus spp., will find use with thepresent expression constructs. Yeast hosts useful in the presentinvention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosacchcromyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Nucleic acid molecules comprising nucleotide sequences of interest canbe stably integrated into a host cell genome or maintained on a stableepisomal element in a suitable host cell using various gene deliverytechniques well known in the art.

Depending on the expression system and host selected, the molecules areproduced by growing host cells transformed by an expression vectordescribed above under conditions whereby the protein is expressed. Theexpressed protein is then isolated from the host cells and purified. Ifthe expression system secretes the protein into growth media, theproduct can be purified directly from the media. If it is not secreted,it can be isolated from cell lysates. The selection of the appropriategrowth conditions and recovery methods are within the skill of the art.

For representative methods for obtaining CEV virus sequencesrecombinantly, see, e.g., Pekosz et al. 1995 J Virol 69:3475-3481. Onceproduced, the various polypeptides and polynucleotides can be formulatedinto subunit vaccine compositions for use as prophylactics ortherapeutics, or used in diagnostic assays, as described below.

Inactivated CEV Serogroup Vaccines

Aspects of the invention also include compositions comprisinginactivated (or killed) viruses of the CEV serogroup, such asinactivated LACV, and methods for the production thereof Inactivatedviral compositions can be used as prophylactic or therapeutic vaccines.Preferably the inactivated vaccine compositions comprise an amount ofinactivated virus equivalent to a virus titer of from about 10³ to 10¹²plaque forming units (PFU) or 10³ to 10¹² tissue culture infectious dose50 (TCID₅₀) per milliliter, preferably 10⁴ to 10¹⁰ PFU or TCID₅₀ evenmore preferably from about 10⁵ to 10⁹ PFU or TCID₅₀ per milliliter, orany dose within these stated ranges. The vaccine compositions comprise asufficient amount of the virus antigen to produce an immunologicalresponse in a mammal, as defined above. Such compositions are describedmore fully below.

Virus can be obtained directly from the ATCC as described above. Othersources of virus include plasma, serum, or tissue homogenates from CEVserogroup infected individuals. Alternatively, virus can be isolatedfrom infected mosquitos, or can be genetically modified or chimerized.Once obtained, the virus can be propagated using known techniques, suchas described in Pekosz et al. 1995 J Virol 69:3475-3481. Viruses of theCEV serogroup are generally cultured in either an adherent or suspensionmammalian cell culture.

Methods of inactivating or killing viruses are known in the art. Suchmethods destroy the ability of the viruses to infect mammalian cells.Inactivation can be achieved using either chemical or physical means.Chemical means for inactivating a virus include treatment of the viruswith an effective amount of one or more of the following agents:detergents, formaldehyde, formalin, 3-propiolactone, or UV light. Othermethods of viral inactivation are known in the art, such as for examplebinary ethylamine, acetyl ethyleneimine, or gamma irradiation.

Attenuated CEV Serogroup Vaccines

Aspects of the invention also include compositions (e.g., immunogeniccompositions) comprising, consisting of, or consisting essentially ofattenuated viruses of the CEV serogroup. As used herein, attenuationrefers to the decreased virulence of the CEV virus in a mammalian host.The compositions can be used as prophylactics or therapeutics. Methodsof attenuating viruses are known in the art. Such methods include serialpassage of the virus in cultured cells as described above (e.g.,mammalian cell culture, preferably BHK or VERO cells), until the virusdemonstrates attenuated function. The temperature at which the virus isgrown can be any temperature at which tissue culture passage attenuationoccurs. Attenuated function of the virus after one or more passages incell culture can be measured by one skilled in the art. Evidence ofattenuated function may be indicated by decreased levels of viralreplication or by decreased virulence in an animal model. Acceptableanimal models for studying viruses of the CEV serogroup are known in theart.

Other methods of producing an attenuated viruses from the CEV serogroupinclude passage of the virus in cell culture at suboptimal or “cold”temperatures and/or introduction of attenuating mutations into the viralgenome by random mutagenesis (e.g., chemical mutagenesis) orsite-directed mutagenesis.

The attenuated virus derivatives of the CEV serogroup are produced inseveral ways, such as for example, by introduction of temperaturesensitive (ts) mutations either with or without chemical mutagenesis(e.g., 5-fluorouracil), by passage in culture at “cold” temperatures.Such cold adaptation includes passage at temperatures between about 20°C. to about 32° C., and preferably between temperatures of about 22° C.to about 30° C., and most preferably between temperatures of about 24°C. and 28° C. The cold adaptation or attenuation may be performed bypassage at increasingly reduced temperatures to introduce additionalgrowth restriction mutations. The number of passages required to obtainsafe, immunizing attenuated virus is dependent at least in part on theconditions employed. Periodic testing of the virus culture for virulenceand immunizing ability in animals (e.g., mouse, primate) can readilydetermine the parameters for a particular combination of tissue cultureand temperature. The attenuated vaccine and/or immunogenic compositionwill typically be formulated in a dose of from about 10² to 10¹² PFU or10² to 10¹² tissue culture infectious dose 50 (TCID₅₀) per milliliter,preferably 10⁴ to 10¹⁰ PFU or TCID₅₀, even more preferably from about10⁵ to 10⁹ PFU or TCID₅₀ per milliliter, or any dose within these statedranges.

Viruses of the CEV serogroup can also be attenuated by mutating one ormore of the various viral regions to reduce expression of the viralstructural or nonstructural proteins. The attenuated CEV may comprise,consist, or consist essentially of one or more additions, deletions orinsertions in one or more of the regions of the viral genome. Forexample, the hydrophobic domains of CEV serogroup proteins are targetsfor genetic mutation to develop attenuated CEV serogroup vaccines. Thehydrophobic domains are also targets for small molecule inhibitors ofCEV viruses. The hydrophobic domains may also be used to generateantibodies specific to those regions to treat or prevent CEV serogroupinfection.

Transmembrane and hydrophobic regions of the CEV serogroup proteins arereadily identified using programs well known in the art, such as theKyte-Doolittle technique.

In one embodiment, the virus is attenuated by ablation at thetranslational level without deletion of the gene or of a segmentthereof, by, e.g., introducing a translational termination codon into atranslational open reading frame (ORF) (e.g., the rLACVdelNSs mutation(Blakqori, G. and Weber F. 2005 J Virol 79:10420-10428). Blakqori andWeber generated a mutant virus with an inactivated NSs gene. Because theN and NSs reading frames overlap, complete deletion of the NSs gene isnot possible without affecting the N gene. Referring to Blakqori andWeber, two tandem ATG start codons of NSs were changed to ACG and theadjacent, downstream codon was changed to a stop codon. The resultingantigenomic LACV S construct expresses an unaltered N protein but noNSs. Infection with bunyaviruses is known to induce a strong shutoff ofhost cell protein synthesis. The rLACVdelNSs virus, by contrast,displayed weaker shutoff. Thus, the rLACVdelNSs mutation attenuates thevirus.

Compositions Comprising CEV Serogroup, Polypeptides, and Polynucleotides

Aspects of the invention provide compositions including theabove-described viruses of the CEV serogroup (e.g., inactivated andattenuated), as well as CEV serogroup polypeptides and/orpolynucleotides. Some embodiments may comprise a pharmaceuticallyacceptable carrier. The carrier should not itself induce the productionof antibodies harmful to the host. Pharmaceutically acceptable carriersare well known to those in the art. Such carriers include, but are notlimited to, large, slowly metabolized, macromolecules, such as proteins,polysaccharides such as latex functionalized sepharose, agarose,cellulose, cellulose beads and the like, polylactic acids, polyglycolicacids, polymeric amino acids such as polyglutamic acid, polylysine, andthe like, amino acid copolymers, and inactive virus particles.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art. Someembodiments can also contain liquids or excipients, such as water,saline, glycerol, dextrose, ethanol, or the like, singly or incombination, as well as, substances such as wetting agents, emulsifyingagents, or pH buffering agents. Liposomes can also be used as a carrierfor a composition of the invention and are described below.

If desired, co-stimulatory molecules which improve immunogenpresentation to lymphocytes, such as B7-1 or B7-2, or cytokines such asGM-CSF, IL-2, and IL-12, can be included.

Optionally, adjuvants can also be included in a composition. Adjuvantswhich can be used include, but are not limited to: (1) aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, aluminumsulfate, etc.; (2) oil-in-water emulsion formulations (with or withoutother specific immunostimulating agents; (3) saponin adjuvants, such asQS21 may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) andIncomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, interferons (e.g., gammainterferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) detoxified mutants of a bacterialADP-ribosylating toxin such as a cholera toxin (CT), a pertussis toxin(PT), or an E. coli heat-labile toxin (LT); (7) MPL or 3-O-deacylatedMPL (3dMPL); (8) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions; (9) oligonucleotides comprising CpG motifs; (10)a polyoxyethylene ether or a polyoxyethylene ester; (11) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol or a polyoxyethylene alkyl ether or ester surfactant incombination with at least one additional non-ionic surfactant such as anoctoxynol; (12) a saponin and an immunostimulatory oligonucleotide suchas a CpG oligonucleotide; (13) an immunostimulant and a particle ofmetal salt; and (14) other substances that act as immunostimulatingagents to enhance the effectiveness of the composition.

Some embodiments may comprise, consist of or consist essentially of atherapeutically effective amount of the desired CEV serogroup moleculeor inactivated or attenuated viruses of the CEV serogroup and any otherof the above-mentioned components, as needed. By “therapeuticallyeffective amount” is meant an amount of a protein or DNA encoding thesame which will induce an immunological response, preferably aprotective immunological response, in the individual to which it isadministered, if the composition is to be used as a vaccine. Such aresponse will generally result in the development in the subject of anantibody-mediated and/or a secretory or cellular immune response to thecomposition. Usually, such a response includes but is not limited to oneor more of the following effects; the production of antibodies from anyof the immunological classes, such as immunoglobulins A, D, E, G or M;the proliferation of B and T lymphocytes; the provision of activation,growth and differentiation signals to immunological cells; expansion ofhelper T cell, suppressor T cell, and/or cytotoxic T cell and/or γδTcell populations.

Administration

The immunogenic compositions (both DNA and protein) can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. Thus, once formulated, the compositionsare conventionally administered parenterally, e.g., by injection, eithersubcutaneously or intramuscularly. For example, the immunogen ispreferably administered intramuscularly to a large mammal, such as aprimate, for example, a baboon, chimpanzee, or human. Additionalformulations suitable for other modes of administration include oral andpulmonary formulations, suppositories, and transdermal formulations,aerosol, intranasal, oral formulations, and sustained releaseformulations. For example, the immunogenic compositions described hereincan be provided as DNA immunogens (e.g., DNA vaccination) intradermally,intramuscularly, or intranasally. (See e.g., Intervirology 43: 312321(2000) and Human Gene Therapy 10:1649-1658 (1999), herein expresslyincorporated by reference in its entirety).

In one set of experiments, an immunogenic composition prepared asdescribed herein (e.g., LACV/human/1960) was provided to three-week oldSwiss Webster weanling mice (n=6/dose) intranasally (IN) in 10 ul volumeor intraperitoneally (IP) in 100 ul volume in serial dilutions. The 50%lethal dose (LD50) and 50% infectious dose (ID50) were determined. Inboth groups, clinical disease was first noted on day 6. Twenty dayspost-inoculation, the LD50 was determined and the mice were tested forthe development of a neutralizing antibody response. The LD50 for bothIN and IP groups were similar (2.4 and 2.3 log 10 PFU, respectively). Todetermine the ID50 titer, mice were considered infected if they eitherdeveloped clinical disease or a serum neutralizing antibody titer. TheID50 titers (1.5 and 1.6 log 10 PFU for the IN and IP routes,respectively) were slightly lower than the LD50 titers indicating thatLACV can cause subclinical infection in weanling mice at low doses.

Controlled or sustained release formulations are made by incorporatingthe active agent into carriers or vehicles such as liposomes,nonresorbable impermeable polymers such as ethylenevinyl acetatecopolymers, swellable polymers such as hydrogels, or resorbable polymerssuch as collagen and certain polyacids or polyesters such as those usedto make resorbable sutures. The immunogens can also be delivered usingimplanted mini-pumps, well known in the art.

The immunogens described herein can also be provided via a carrier viruswhich expresses the same. Carrier viruses which will find use with theinstant invention include but are not limited to the vaccinia and otherpox viruses, adenovirus, and herpes virus. By way of example, vacciniavirus recombinants expressing the novel proteins can be constructed asfollows. The DNA encoding the particular protein is first inserted intoan appropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the instantprotein into the viral genome. The resulting TK″ recombinant can beselected by culturing the cells in the presence of 5-bromodeoxyuridineand picking viral plaques resistant thereto.

The immunogens can be provided either to a mammal that is not infectedwith a virus of the CEV serogroup or can be administered to aCEV-infected mammal.

Dosage treatment may be a single dose schedule or a multiple doseschedule. Preferably, the effective amount is sufficient to bring abouttreatment or prevention of disease symptoms. The exact amount necessarywill vary depending on the subject being treated, the age and generalcondition of the individual to be treated; the capacity of theindividual's immune system to synthesize antibodies; the degree ofprotection desired; the severity of the condition being treated; theparticular macromolecule selected and its mode of administration, amongother factors. An appropriate effective amount can be readily determinedby one of skill in the art. A “therapeutically effective amount” willfall in a relatively broad range that can be determined through routinetrials using in vitro and in vivo models known in the art.

Thus, for example, if polypeptide immunogens are delivered, generallythe amount administered will be about 0.1 μg to about 750 μg ofimmunogen per dose, or any amount between the stated ranges, such as 1μg to about 500 μg, 5 μg to about 250 μg, 10 μg to about 100 μg, 10 μgto about 50 μg, such as 4, 5, 6, 7, 8, 10, 20, 25, 30, 35, 40, 50, 60,70, 80, 90, 100, etc., μg per dose.

As explained above, expression constructs, such as constructs encodingindividual CEV serogroup immunogens or fusions, may be used for nucleicacid immunization to stimulate an immunological response, such as acellular immune; response and/or humoral immune response, using standardgene delivery protocols. Methods for gene delivery are known in the art.Genes can be delivered either directly to the subject or, alternatively,delivered ex vivo, to cells derived from the subject and the cellsreimplanted in the subject. For example, the constructs can be deliveredas plasmid DNA, e.g., contained within a plasmid, such as pBR322, pUC,or ColE1.

Additionally, the expression constructs can be packaged in liposomesprior to delivery to the cells. Lipid encapsulation is generallyaccomplished using liposomes which are able to stably bind or entrap andretain nucleic acid. The ratio of condensed DNA to lipid preparation canvary but will generally be around 1:1 (mg DNA:micromoles lipid), or moreof lipid.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems, such as murine sarcoma virus, mousemammary tumor virus, Moloney murine leukemia virus, and Rous sarcomavirus. A selected gene can be inserted into a vector and packaged inretroviral particles using techniques known in the art. The recombinantvirus can then be isolated and delivered to cells of the subject eitherin vivo or ex vivo. A number of retroviral systems have been describedin the scientific and patent literature. Briefly, retroviral genedelivery vehicles of the present invention may be readily constructedfrom a wide variety of retroviruses, including for example, B, C, and Dtype retroviruses as well as spumaviruses and lentiviruses such as FIV,HIV, HIV-1, HIV-2 and SIV. Such retroviruses may be readily obtainedfrom depositories or collections such as the American Type CultureCollection (“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209),or isolated from known sources using commonly available techniques.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are useful for delivering the expressionconstructs of the present invention. The particles are coated with theconstruct to be delivered and accelerated to high velocity, generallyunder a reduced atmosphere, using a gun powder discharge from a “genegun”.

The amount of CEV serogroup DNA delivered will generally be about 1 μgto 500 mg of DNA (e.g., at least or equal to 5 μg to 100 mg of DNA,e.g., 10 μg to 50 mg, or 100 μg to 5 mg, such as 20, 30, 40, 50, 60,100, 200 μg and so on, to 500 μg DNA, and any integer between the statedranges).

Administration of CEV serogroup virions, polypeptide or polynucleotidecompositions can elicit a cellular immune response, and/or an anti-CEVserogroup antibody titer in the mammal that lasts for at least 1 week, 2weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 1 year, orlonger. The compositions can also be administered to provide a memoryresponse. If such a response is achieved, antibody titers may declineover time, however exposure to virus or the particular immunogen resultsin the rapid induction of antibodies, e.g., within only a few days.Optionally, antibody titers can be maintained in a mammal by providingone or more booster injections of the compositions, at e.g., 2 weeks, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or moreafter the primary injection.

Preferably, an antibody titer of at least 10, 100, 150, 175, 200, 300,400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000, 10,000, 20,000,30,000, 40,000, 50,000 (geometric mean titer), or higher, is elicited,air any number between the stated titers, as determined using a standardimmunoassay.

CEV Serogroup Antibodies

The CEV serogroup immunogens can be used to produce CEV-specificpolyclonal and monoclonal antibodies. CEV-specific polyclonal andmonoclonal antibodies specifically bind to CEV serogroup antigens.Polyclonal antibodies can be produced by administering the immunogen toa mammal, such as a mouse, a rabbit, a goat, or a horse. Serum from theimmunized animal is collected and the antibodies are purified from theplasma by, for example, precipitation with ammonium sulfate, followed bychromatography, preferably affinity chromatography. Techniques forproducing and processing polyclonal antisera are known in the art.

Monoclonal antibodies directed against CEV serogroup-specific epitopespresent in the proteins can also be readily produced. Normal B cellsfrom a mammal, such as a mouse, immunized with a CEV serogroup protein,can be fused with, for example, HAT-sensitive mouse myeloma cells toproduce hybridomas. Hybridomas producing CEV serogroup-specificantibodies can be identified using RIA or ELISA and isolated by cloningin semi-solid agar or by limiting dilution. Clones producing CEVserogroup-specific antibodies are isolated by another round ofscreening.

It may be desirable to provide chimeric antibodies, especially if theantibodies are to be used in preventive or therapeutic pharmaceuticalpreparations, such as for providing passive protection against CEVserogroup infection, as well as in CEV serogroup diagnosticpreparations. Chimeric antibodies composed of human and non-human aminoacid sequences may be formed from the mouse monoclonal antibodymolecules to reduce their immunogenicity in humans.

Antibody molecule fragments, e.g., F(ab′)2, Fv, and sFv molecules, thatare capable of exhibiting immunological binding properties of the parentmonoclonal antibody molecule can be produced using known techniques. Inthe alternative, a phage-display system can be used to expand monoclonalantibody molecule populations in vitro.

Once generated, the phage display library can be used to improve theimmunological binding affinity of the Fab molecules using knowntechniques. The coding sequences for the heavy and light chain portionsof the Fab molecules selected from the phage display library can beisolated or synthesized, and cloned into any suitable vector or repliconfor expression. Any suitable expression system can be used, includingthose described above.

Antibodies which are directed against CEV serogroup epitopes areparticularly useful for detecting the presence of CEV or viral antigensin a sample, such as a serum sample from a CEV serogroup-infected human.An immunoassay for a CEV serogroup antigen may utilize one antibody orseveral antibodies. An immunoassay for a CEV serogroup antigen may use,for example, a monoclonal antibody directed towards a CEV serogroupepitope, a combination of monoclonal antibodies directed towardsepitopes of one CEV serogroup polypeptide, monoclonal antibodiesdirected towards epitopes of different CEV serogroup polypeptides,polyclonal antibodies directed towards the same CEV serogroup antigen,polyclonal antibodies directed towards different CEV serogroup antigens,or a combination of monoclonal and polyclonal antibodies. Immunoassayprotocols may be based, for example, upon competition, direct reaction,or sandwich type assays using, for example, labeled antibody and aredescribed further below. The labels may be, for example, fluorescent,chemiluminescent, or radioactive.

The CEV serogroup antibodies may further be used to isolate CEVserogroup particles or antigens by immunoaffinity columns. Theantibodies can be affixed to a solid support by, for example, adsorptionor by covalent linkage so that the antibodies retain theirimmunoselective activity. Optionally, spacer groups may be included sothat the antigen binding site of the antibody remains accessible. Theimmobilized antibodies can then be used to bind CEV serogroup particlesor antigens from a biological sample, such as blood or plasma. The boundCEV serogroup particles or antigens are recovered from the column matrixby, for example, a change in pH.

CEV Serogroup Diagnostic Assays

As explained above, the CEV serogroup immunogens, antibodies andpolynucleotides can be used in assays to identify CEV serogroupinfection, such as LACV infection. Protein assays include Western blots;agglutination tests; enzyme-labeled and mediated immunoassays, such asELISAs; biotin/avidin type assays; radioimmunoassays,immunoelectrophoresis; immunoprecipitation, and the like. The reactionsgenerally include revealing labels such as fluorescent,chemiluminescent, radioactive, enzymatic labels or dye molecules, orother methods for detecting the formation of a complex between themimetic and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unboundantibody or antigen in a liquid phase from a solid phase support towhich antigen-antibody complexes are bound. Solid supports which can beused in the practice of some embodiments include substrates or supportssuch as nitrocellulose (e.g., in membrane or microtiter well form);polyvinylchloride (e.g., sheets or microtiter wells), polystyrene latex(e.g., beads or microtiter plates), polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

Typically, a solid support is first reacted with a solid phase component(e.g., one or more CEV serogroup viral antigens or antibodies) undersuitable binding conditions such that the component is sufficientlyimmobilized to the support. Sometimes, immobilization to the support canbe enhanced by first coupling to a protein with better bindingproperties. Suitable coupling proteins include, but are not limited to,macromolecules such as serum albumins including bovine serum albumin(BSA), keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Other molecules that can be used to bind the antigen orantibody to the support include polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andthe like. Such molecules and methods of coupling these molecules arewell known to those of ordinary skill in the art.

After reacting the solid support with the solid phase component, anynon-immobilized solid-phase components are removed from the support bywashing, and the support-bound component is then contacted with abiological sample suspected of containing the ligand component (i.e.,CEV serogroup antigens or antibodies) under suitable binding conditions.After washing to remove any non-bound ligand, a secondary binder moietyis added under suitable binding conditions, wherein the secondary binderis capable of associating selectively with the bound ligand. Thepresence of the secondary binder can then be detected using techniqueswell known in the art.

More particularly, an ELISA method can be used, wherein the wells of amicrotiter plate are coated with one or more CEV serogroup epitopes orantibodies according to the present invention. A biological samplecontaining or suspected of containing either anti-CEV virusimmunoglobulin molecules or CEV serogroup antigens is then added to thecoated wells. After a period of incubation sufficient to allowantigen-antibody binding, the plate(s) can be washed to remove unboundmoieties and a detectably labeled secondary binding molecule added. Thesecondary binding molecule is allowed to react with any captured sample,the plate washed and the presence of the secondary binding moleculedetected using methods well known in the art.

Thus, in one particular embodiment, the presence of bound CEV serogroupligands from a biological sample can be readily detected using asecondary binder comprising an antibody directed against the antibodyligands. A number of anti-human immunoglobulin (Ig) molecules are knownin the art which can be readily conjugated to a detectable enzyme label,such as horseradish peroxidase, alkaline phosphatase or urease, usingmethods known to those of skill in the art. An appropriate enzymesubstrate is then used to generate a detectable signal. In other relatedembodiments, competitive-type ELISA techniques can be practiced usingmethods known to those skilled in the art.

Assays can also be conducted in solution, such that the CEV serogroupepitopes or antibodies and ligands specific for these molecules formcomplexes under precipitating conditions. In one particular embodiment,the molecules can be attached to a solid phase particle (e.g., anagarose bead or the like) using coupling techniques known in the art,such as by direct chemical or indirect coupling. The coated particle isthen contacted under suitable binding conditions with a biologicalsample suspected of containing CEV serogroup antibodies or antigens.Cross-linking between bound antibodies causes the formation of complexaggregates which can be precipitated and separated from the sample usingwashing and/or centrifugation. The reaction mixture can be analyzed todetermine the presence or absence of complexes using any of a number ofstandard methods, such as those immunodiagnostic methods describedabove.

In yet a further embodiment, an immunoaffinity matrix can be provided,wherein, for example, a polyclonal population of antibodies from abiological sample suspected of containing CEV serogroup antibodies isimmobilized to a substrate. An initial affinity purification of thesample can be carried out using immobilized antigens. The resultantsample preparation will thus only contain anti-CEV serogroup moieties,avoiding potential nonspecific binding properties in the affinitysupport. A number of methods of immobilizing immunoglobulins (eitherintact or in specific fragments) at high yield and good retention ofantigen binding activity are known in the art. Once the immunoglobulinmolecules have been immobilized to provide an immunoaffinity matrix,labeled molecules are contacted with the bound antibodies under suitablebinding conditions. After any non-specifically bound CEV serogroupepitope has been washed from the immunoaffinity support, the presence ofbound antigen can be determined by assaying for label using methodsknown in the art.

The above-described assay reagents, including CEV serogroup polypeptidesand/or antibodies thereto, the solid supports with bound reagents, aswell as other detection reagents, can be provided in kits, with suitableinstructions and other necessary reagents, in order to conduct theassays as described above. The kit may also include control formulations(positive and/or negative), labeled reagents when the assay formatrequires same and signal generating reagents (e.g., enzyme substrate) ifthe label does not generate a signal directly. Instructions (e.g.,written, tape, VCR, CD ROM, etc.) for carrying out the assay usuallywill be included in the kit. The kit can also contain, depending on theparticular assay used, other packaged reagents and materials (i.e.,buffers and the like). Standard assays, such as those described above,can be conducted using these kits.

Nucleic acid-based assays can be conducted using CEV serogroupoligonucleotides and polynucleotides described above. For example,probe-based assays, such as hybridization assays, can be conducted thatutilize oligonucleotides from the specific virus in question. Theseassays may also utilize nucleic acid amplification methods such asreverse transcriptase-polymerase chain reaction (RT-PCR), PCR and ligasechain reaction (LCR).

Thus, the various CEV serogroup polynucleotide sequences may be used toproduce probes and primers which can be used in assays for the detectionof nucleic acids in test samples. The probes and primers may be designedfrom conserved nucleotide regions of the polynucleotides of interest orfrom non-conserved nucleotide regions of the polynucleotide of interest.The design of such oligonucleotides is routine in the art. Generally,nucleic acid probes are developed from non-conserved or unique regionswhen maximum specificity is desired, and nucleic acid probes aredeveloped from conserved regions when assaying for nucleotide regionsthat are closely related to, for example, different CEV serogroupisolates.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence.By selection of appropriate conditions, the probe and the targetsequence “selectively hybridize”, or bind to each other to form a hybridmolecule. An oligonucleotide that “selectively hybridizes” to a LACVsequence under hybridization conditions described below, denotes anoligonucleotide, e.g., a primer, probe or a capture oligonucleotide,that binds to a LACV sequence but does not bind to a sequence from otherviruses of the CEV serogroup. In one embodiment of the presentinvention, a nucleic acid molecule is capable of hybridizing selectivelyto a target sequence under moderately stringent hybridizationconditions. In the context of the present invention, moderatelystringent hybridization conditions allow detection of a target nucleicacid sequence of at least 14 nucleotides in length having at leastapproximately 70% sequence identity with the sequence of the selectednucleic acid probe. In another embodiment, such selective hybridizationis performed under stringent hybridization conditions. Stringenthybridization conditions allow detection of target nucleic acidsequences of at least 14 nucleotides in length having a sequenceidentity of greater than 90% with the sequence of the selected nucleicacid probe. Hybridization conditions useful for probe/targethybridization where the probe and target have a specific degree ofsequence identity, can be determined as is known in the art. Hybridmolecules can be formed, for example, on a solid support, in solution,and in tissue sections. The formation of hybrids can be monitored byinclusion of a reporter molecule, typically, in the probe. Such reportermolecules, or detectable elements include, but are not limited to,radioactive elements, fluorescent markers, and molecules to which anenzyme-conjugated ligand can bind.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is well known (see, forexample, Sambrook, et al., supra.)

As explained above, the primers and probes may be used in polymerasechain reaction (PCR)-based techniques, such as RT-PCR, to detect CEVserogroup infection in biological samples. PCR is a technique foramplifying a desired target nucleic acid sequence contained in a nucleicacid molecule or mixture of molecules. In PCR, a pair of primers isemployed in excess to hybridize to the complementary strands of thetarget nucleic acid. The primers are each extended by a polymerase usingthe target nucleic acid as a template. The extension products becometarget sequences themselves after dissociation from the original targetstrand. New primers are then hybridized and extended by a polymerase,and the cycle is repented to geometrically increase the number of targetsequence molecules. The PCR method for amplifying target nucleic acidsequences in a sample is well known in the art and has been describedin, e.g., Innis et al. (eds.) PCR Protocols (Academic Press, N Y 1990).

In particular, PCR uses relatively short oligonucleotide primers whichflank the target nucleotide sequence to be amplified, oriented such thattheir 3′ ends face each other, each primer extending toward the other.The polynucleotide sample is extracted and denatured, preferably byheat, and hybridized with first and second primers that are present inmolar excess. Polymerization is catalyzed in the presence of the fourdeoxyribonucleotide triphosphates (dNTPs—dATP, dGTP, dCTP and dTTP)using a primer- and template-dependent polynucleotide polymerizingagent, such as any enzyme capable of producing primer extensionproducts, for example, thermostable DNA polymerases isolated fromTherrmus aquaticus (Taq), available from a variety of sources (forexample, Perkin Elmer). This results in two “long products” whichcontain the respective primers at their 5′ ends covalently linked to thenewly synthesized complements of the original strands. The reactionmixture is then returned to polymerizing conditions, e.g., by loweringthe temperature, inactivating a denaturing agent, or adding morepolymerase, and a second cycle is initiated. The second cycle providesthe two original strands, the two long products from the first cycle,two new long products replicated from the original strands, and two“short products” replicated from the long products. The short productshave the sequence of the target sequence with a primer at each end. Oneach additional cycle, an additional two long products are produced, anda number of short products equal to the number of long and shortproducts remaining at the end of the previous cycle. Thus, the number ofshort products containing the target sequence grows exponentially witheach cycle. Preferably, PCR is carried out with a commercially availablethermal cycler, e.g., Perkin Elmer.

RNAs may be amplified by reverse transcribing the RNA into cDNA, andthen performing PCR (RT-PCR), as described above. Alternatively, asingle enzyme may be used for both steps.

The Ligase Chain Reaction (LCR) is an alternate method for nucleic acidamplification. In LCR, probe pairs are used which include two primary(first and second) and two secondary (third and fourth) probes, all ofwhich are employed in molar excess to target. The first probe hybridizesto a first segment of the target strand, and the second probe hybridizesto a second segment of the target strand, the first and second segmentsbeing contiguous so that the primary probes abut one another in 5′phosphate-3′ hydroxyl relationship, and so that a ligase can covalentlyfuse or ligate the two probes into a fused product. In addition, a third(secondary) probe can hybridize to a portion of the first probe and afourth (secondary) probe can hybridize to a portion of the second probein a similar abutting fashion. If the target is initially doublestranded, the secondary probes also will hybridize to the targetcomplement in the first instance. Once the ligated strand of primaryprobes is separated from the target strand, it will hybridize with thethird and fourth probes which can be ligated to form a complementary,secondary ligated product. It is important to realize that the ligatedproducts are functionally equivalent to either the target or itscomplement. By repeated cycles of hybridization and ligation,amplification of the target sequence is achieved.

The fluorogenic 5′ nuclease assay, known as the TaqMan™ assay(Perkin-Elmer), is a powerful and versatile PCR-based detection systemfor nucleic acid targets. Hence, primers and probes derived fromconserved and/or non-conserved regions of the CEV serogroup genome inquestion can be used in TaqMan™ analyses to detect the presence ofinfection in a biological sample. Analysis is performed in conjunctionwith thermal cycling by monitoring the generation of fluorescencesignals. The assay system dispenses with the need for gelelectrophoretic analysis, and is capable of generating quantitative dataallowing the determination of target copy numbers. For example, standardcurves can be produced using serial dilutions of previously quantifiedCEV serogroup suspensions. A standard graph can be produced with copynumbers of each of the panel members against which sample unknowns canbe compared.

The fluorogenic 5′ nuclease assay is conveniently performed using, forexample, AmpliTaq Gold™ DNA polymerase, which has endogenous 5′ nucleaseactivity, to digest an internal oligonucleotide probe labeled with botha fluorescent reporter dye and a quencher. Assay results are detected bymeasuring changes in fluorescence that occur during the amplificationcycle as the fluorescent probe is digested, uncoupling the dye andquencher labels and causing an increase in the fluorescent signal thatis proportional to the amplification of target nucleic acid.

The amplification products can be detected in solution or using solidsupports. In this method, the TaqMan™ probe is designed to hybridize toa target sequence within the desired PCR product. The 5′ end of theTaqMan™ probe contains a fluorescent reporter dye. The 3′ end of theprobe is blocked to prevent probe extension and contains a dye that willquench the fluorescence of the 5′ fluorophore. During subsequentamplification, the 5′ fluorescent label is cleaved off if a polymerasewith 5′ exonuclease activity is present in the reaction. Excision of the5′ fluorophore results in an increase in fluorescence that can bedetected.

Accordingly, the present invention relates to methods for amplifying atarget CEV serogroup nucleotide sequence using a nucleic acid polymerasehaving 5′ to 3′ nuclease activity, one or more primers capable ofhybridizing to the CEV serogroup target sequence, and an oligonucleotideprobe capable of hybridizing to the CEV serogroup target sequence 3′relative to the primer. During amplification, the polymerase digests theoligonucleotide probe when it is hybridized to the target sequence,thereby separating the reporter molecule from the quencher molecule. Asthe amplification is conducted, the fluorescence of the reportermolecule is monitored, with fluorescence corresponding to the occurrenceof nucleic acid amplification. The reporter molecule is preferably afluorescein dye and the quencher molecule is preferably a rhodamine dye.

While the length of the primers and probes can vary, the probe sequencesare selected such that they have a higher melt temperature than theprimer sequences. Preferably, the probe sequences have an estimated melttemperature that is about 10° C. higher than the melt temperature forthe amplification primer sequences. Hence, the primer sequences aregenerally shorter than the probe sequences. Typically, the primersequences are in the range of between 10-75 nucleotides long, moretypically in the range of 20-45. The typical probe is in the range ofbetween 10-50 nucleotides long, more typically 15-40 nucleotides inlength. Representative primers and probes useful in TaqMan™ assays aredescribed above.

As is readily apparent, design of the assays described herein aresubject to a great deal of variation, and many formats are known in theart. The above descriptions are merely provided as guidance and one ofskill in the art can readily modify the described protocols, usingtechniques well known in the art.

The above-described assay reagents, including the primers, probes, solidsupport with bound probes, as well as other detection reagents, can beprovided in kits, with suitable instructions and other necessaryreagents, in order to conduct the assays as described above. The kitwill normally contain in separate containers the combination of primersand probes (either already bound to a solid matrix or separate withreagents for binding them to the matrix), control formulations (positiveand/or negative), labeled reagents when the assay format requires sameand signal generating reagents (e.g., enzyme substrate) if the labeldoes not generate a signal directly.

Chimeric Attenuated Viruses

Aspects of the present invention provide infectious, chimeric viruses ofthe Bunyaviridae family that are attenuated and capable of eliciting aprophylactic or therapeutic immune response in mammalian patientssusceptible to viral infection. Some embodiments also include methodsand compositions for designing and producing attenuated, chimericviruses of the Bunyaviridae family, as well as methods and compositionsfor prophylaxis and treatment of infection by viruses of theBunyaviridae family.

Chimeric Bunyaviridae embodiments are recombinantly engineered toincorporate nucleotide sequences from more than one virus strain orsubgroup to produce an infectious, chimeric virus or subviral particle.In this manner, candidate vaccine viruses are recombinantly engineeredto elicit an immune response against a virus of the Bunyaviridae familyin a mammalian host, including humans, non-human primates, andlivestock/food animals. Chimeric Bunyaviridae as described herein mayelicit an immune response to a specific Bunyaviridae subgroup or strain,or they may elicit a polyspecific response against multiple Bunyaviridaesubgroups or strains.

In exemplary embodiments, heterologous genes, gene segments, or singleor multiple nucleotides of one virus of the Bunyaviridae family areadded to a partial or complete genome or substituted therein bycounterpart sequence(s) from a heterologous virus of the Bunyaviridaefamily to produce a chimeric genome. The chimeric Bunyaviridae describedherein may include a partial or complete “recipient” Bunyaviridae genomefrom one viral strain or subgroup virus combined with an additional orreplacement “donor” gene or gene segment of a different Bunyaviridaestrain or subgroup virus.

In preferred embodiments, chimeric Bunyaviridae incorporate a partial orcomplete LACV genome combined with a heterologous gene or gene segmentfrom a different Bunyaviridae subgroup or strain. For example, one typeof chimeric Bunyaviridae incorporates a chimeric genome comprised of apartial or complete genome of a LACV combined with a heterologous geneor gene segment from a hantavirus or Jamestown Canyon virus.

Heterologous donor genes or gene segments from one Bunyaviridae orsubgroup are combined with or substituted within a recipient genome thatserves as a genetic background for insertion or addition of the donorgene or gene segment. Thus, the recipient genome acts as a vector toimport and express heterologous genes or gene segments to yield chimericBunyaviridae that exhibit novel structural and/or phenotypiccharacteristics. Preferably, addition or substitution of a heterologousgene or gene segment within a selected recipient Bunyaviridae strainyields novel phenotypic effects, for example attenuation, growthchanges, altered immunogenicity, or other desired phenotypic changes, ascompared with corresponding phenotypes of the unmodified recipientand/or donor.

Genes and gene segments that are useful as heterologous inserts oradditions within a chimeric Bunyaviridae genome include genes or genesegments encoding a L, G_(N), NS_(M), G_(C), N, or NSs protein, or aportion thereof. The chimeric Bunyaviridae may incorporate a genesegment encoding only a portion of a selected protein, for example acytoplasmic domain, transmembrane domain, ectodomain or immunogenicepitope.

Thus, the introduction of heterologous immunogenic proteins, domains andepitopes to produce chimeric Bunyaviridae is particularly useful togenerate novel immune responses in an immunized host. Addition orsubstitution of an immunogenic gene or gene segment from a donorBunyaviridae subgroup or strain within a recipient genome of a differentBunyaviridae subgroup or strain can generate an immune response directedagainst the donor subgroup or strain, the recipient subgroup or strain,or against both the donor and recipient subgroup or strain. To achievethis purpose, chimeric Bunyaviridae may also be constructed that expressa chimeric protein, e.g., an immunogenic protein having a domainspecific to a LACV strain or subgroup fused to the domain of a differentBunyaviridae family member. Other exemplary recombinants of this typemay express duplicate protein regions, such as duplicate immunogenicregions.

Although it is often useful to add or substitute entire genes (includingcis-acting elements and coding regions) within a chimeric genome, it isalso useful to transfer only a portion of a donor gene of interest.Quite commonly, non-coding nucleotides such as cis-acting regulatoryelements and intergenic sequences need not be transferred with the donorgene coding region. In addition, a variety of gene segments provideuseful donor polynucleotides for inclusion within a chimeric genome toexpress chimeric Bunyaviridae having novel and useful properties. Thus,heterologous gene segments may beneficially encode a cytoplasmic tail,transmembrane domain or ectodomain, an epitopic site or region, abinding site or region, an active site or region containing an activesite, etc., of a selected protein from one Bunyaviridae family member.These and other gene segments can be added or substituted for acounterpart gene segment in another member of the Bunyaviridae family ofviruses to yield novel chimeric recombinants, for example recombinantsexpressing a chimeric protein having a cytoplasmic tail and/ortransmembrane domain of one member of the Bunyaviridae family fused toan ectodomain of another Bunyaviridae family member. Useful genomesegments in this regard range from about 15-35 nucleotides in the caseof gene segments encoding small functional domains of proteins, e.g.,epitopic sites, to about 50, 75, 100, 200-500, and 500-1,500 or morenucleotides for gene segments encoding larger domains or proteinregions.

To construct chimeric Bunyaviridae, heterologous genes may be added orsubstituted in whole or in part to a background genome to form achimeric genome. In the case of chimeras generated by substitution, aselected protein or protein region (e.g., a cytoplasmic tail,transmembrane domain or ectodomain, an epitopic site or region, abinding site or region, an active site or region containing an activesite, etc.) from one member of the Bunyaviridae family is substitutedfor a counterpart gene or gene segment in a different Bunyaviridaefamily member genome to yield novel recombinants having desiredphenotypic changes compared to wild-type or parent Bunyaviridae familystrains. As used herein, “counterpart” genes, gene segments, proteins orprotein regions refers to two counterpart polynucleotides from aheterologous source, including different genes in a single Bunyaviridaestrain, or different variants of the same gene, including species andallelic variants among different Bunyaviridae family subgroups orstrains.

Counterpart genes and gene segments share at least moderate structuralsimilarity. For example counterpart gene segments may encode a commonstructural domain of a protein of interest. Typically, they will share acommon biological function as well. For example, protein domains encodedby counterpart gene segments may provide a common membrane spanningfunction, a specific binding activity, an immunological recognitionsite, etc. Typically, a desired biological activity shared between theproducts of counterpart genes and gene segments will be substantiallysimilar in quantitative terms, i.e., they will not differ by more than30%, preferably by no more than 20%, more preferably by no more than5-10%.

Counterpart genes and gene segments for use within the invention embracean assemblage of alternate species having a range of size and sequencevariation. However, selection of counterpart genes and gene segmentsrelies on substantial sequence identity between the subjectcounterparts. In this context, a selected polynucleotide “referencesequence” is defined as a sequence or portion thereof present in eitherthe donor or recipient genome. This reference sequence is used as adefined sequence to provide a rational basis for a sequence comparison.For example, the reference sequence may be a defined segment of a cDNAor gene, or a complete cDNA or gene sequence.

Generally, a reference sequence for use in defining counterpart genesand gene segments is at least 20 nucleotides in length, frequently atleast 25 nucleotides in length, and often at least 50 nucleotides inlength. Since two polynucleotides may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide sequence) that issimilar between the two polynucleotides, and (2) may further comprise asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 20 contiguous nucleotide positionswherein a polynucleotide sequence may be compared to a referencesequence of at least 20 contiguous nucleotides and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) of 20 percent or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Optimal alignment ofsequences for aligning a comparison window may be conducted by the localhomology algorithm of Smith & Waterman 1981 Adv Appl Math 2:482, by thehomology alignment algorithm of Needleman & Wunsch 1970 J Mol Biol48:443, by the search for similarity method of Pearson & Lipman 1988Proc Natl Acad Sci USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by inspection, and the best alignment (i. e.,resulting in the highest percentage of sequence similarity over thecomparison window) generated by the various methods is selected.

The term “sequence identity” as used herein means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence.

In addition to these polynucleotide sequence relationships, proteins andprotein regions encoded by chimeric Bunyaviridae as described herein arealso typically selected to have conservative relationships, i.e., tohave substantial sequence identity or sequence similarity, with selectedreference polypeptides. As applied to polypeptides, the term “sequenceidentity” means peptides share identical amino acids at correspondingpositions. The term “sequence similarity” means peptides have identicalor similar amino acids (i.e., conservative substitutions) atcorresponding positions. The term “substantial sequence identity” meansthat two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 90 percent sequenceidentity, more preferably at least 95 percent sequence identity or more(e.g., 99 percent sequence identity). The term “substantial similarity”means that two peptide sequences share corresponding percentages ofsequence similarity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a conservative group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Stereoisomers (e.g., D-amino acids) of the twentyconventional amino acids, unnatural amino acids such asα,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,ω-N-methylarginine, and other amino and imino acids (e.g.,4-hydroxyproline). Moreover, amino acids may be modified byglycosylation, phosphorylation and the like.

Aspects of the invention disclosed herein also concern cDNA-basedmethods that are used to construct a large panel of recombinant,chimeric Bunyaviridae viruses and subviral particles. These recombinantconstructs offer improved characteristics of attenuation andimmunogenicity for use as vaccine agents. Among desired phenotypicchanges in this context are resistance to reversion from an attenuatedphenotype, improvements in attenuation in culture or in a selected hostenvironment, immunogenic characteristics (e.g., as determined byenhancement, or diminution, of an elicited immune response),upregulation or downregulation of transcription and/or translation ofselected viral products, etc.

In preferred embodiments, attenuated, chimeric Bunyaviridae are producedin which the chimeric genome is further modified by introducing one ormore attenuating point mutations that specifies an attenuatingphenotype. These point mutations may be generated de novo and tested forattenuating effects according to a rational design mutagenesis strategy.Alternatively, the attenuating point mutations are identified inbiologically-derived mutant Bunyaviridae family member and thereafterincorporated into a chimeric Bunyaviridae as described herein.

Attenuating point mutations in biologically-derived Bunyaviridae familymember for incorporation within a chimeric vaccine strain may occurnaturally or may be introduced into wild-type Bunyaviridae family memberstrains by well-known mutagenesis procedures. For example, incompletelyattenuated parental Bunyaviridae family member strains can be producedby chemical mutagenesis during virus growth in cell cultures to which achemical mutagen has been added, by selection of virus that has beensubjected to passage at suboptimal temperatures in order to introducegrowth restriction mutations, or by selection of a mutagenized virusthat produces small plaques (sp) in cell culture, as generally describedherein.

By “biologically-derived Bunyaviridae family member” is meant anyBunyaviridae family member not produced by recombinant means. Thus,biologically-derived Bunyaviridae family members include naturallyoccurring Bunyaviridae family members of all subgroups and strains,including, e.g., a naturally occurring Bunyaviridae family member havinga wild-type genomic sequence and a Bunyaviridae family member havinggenomic variations from a reference wild-type virus sequence, e.g., aBunyaviridae family member having a mutation specifying an attenuatedphenotype. Likewise, biologically-derived Bunyaviridae family membersinclude Bunyaviridae family member mutants derived from a parentalBunyaviridae family member strain by, inter alia, artificial mutagenesisand selection procedures.

To produce a satisfactorily attenuated Bunyaviridae family member frombiologically-derived strains, mutations are preferably introduced into aparental strain, which has been incompletely or partially attenuated.Using such partially attenuated strains, additional mutation(s) can begenerated that further attenuate the strain, e.g., to a desired level ofrestricted replication in a mammalian host, while retaining sufficientimmunogenicity to confer protection in vaccinees.

As noted above, production of a sufficiently attenuatedbiologically-derived Bunyaviridae family member mutant can beaccomplished by several known methods. One such procedure involvessubjecting a partially attenuated virus to passage in cell culture atprogressively lower, attenuating temperatures. For example, whereaswild-type virus is typically cultivated at about 34-37° C., thepartially attenuated mutants are produced by passage in cell cultures atsuboptimal temperatures, e.g., 20-26° C. This selection of mutant virusduring cold-passage (cp) substantially eliminates any residual virulencein the derivative strains as compared to the partially attenuatedparent.

Alternatively, specific mutations can be introduced intobiologically-derived Bunyaviridae family member by subjecting apartially attenuated parent virus to chemical mutagenesis, e.g., tointroduce temperature sensitive (ts) mutations or, in the case ofviruses which are already ts, additional ts mutations sufficient toconfer increased attenuation and/or stability of the ts phenotype on theattenuated derivative. Means for the introduction of ts mutations into aBunyaviridae family member include replication of the virus in thepresence of a mutagen such as 5-fluorouridine or 5-fluorouracil in aconcentration of about 10⁻³ to 10⁻⁵ M, preferably about 10⁻⁴M, exposureof virus to nitrosoguanidine at a concentration of about 100 μg/ml,according to the general procedures, or genetic introduction of specificts mutations. Other chemical mutagens can also be used. Attenuation canresult from a ts mutation in almost any Bunyaviridae family member gene.

Mutations thus identified are compiled into a “menu” and are thenintroduced as desired, singly or in combination, to calibrate a chimericvaccine virus to an appropriate level of attenuation, immunogenicity,genetic resistance to reversion from an attenuated phenotype, etc., asdesired. Preferably, chimeric Bunyaviridae as described herein areattenuated by incorporation of at least one, and more preferably two ormore, attenuating point mutations identified from such a menu, which maybe defined as a group of known mutations within a panel ofbiologically-derived mutant Bunyaviridae strains.

Chimeric Bunyaviridae designed and selected for vaccine and immunogeniccomposition use often have at least two and sometimes three or moreattenuating mutations to achieve a satisfactory level of attenuation forbroad clinical use. In one embodiment, at least one attenuating mutationoccurs in the G_(C) or G_(N) gene (either in the donor or recipientgene) and involves a nucleotide substitution specifying an amino acidchange in the G_(C) or G_(N) protein.

In accordance with the methods described herein, chimeric Bunyaviridaecan be readily constructed and characterized that incorporate at leastone and up to a full complement of attenuating point mutations presentwithin a panel of biologically-derived mutant Bunyaviridae strains.Thus, mutations can be assembled in any combination from a selectedpanel of mutants.

In accordance with the foregoing description, the ability to produceinfectious Bunyaviridae from cDNA permits introduction of specificengineered changes within chimeric Bunyaviridae. In particular,infectious, recombinant Bunyaviridae are employed for identification ofspecific mutation(s) in biologically-derived, attenuated Bunyaviridaestrains, for example mutations which specify ts and other phenotypes.Desired mutations are thus identified and introduced into recombinant,chimeric Bunyaviridae vaccine strains. The capability of producing virusfrom cDNA allows for routine incorporation of these mutations,individually or in various selected combinations, into a full-lengthcDNA clone, whereafter the phenotypes of rescued recombinant virusescontaining the introduced mutations can be readily determined.

By identifying and incorporating specific, biologically-derivedmutations associated with desired phenotypes into infectious chimericBunyaviridae clones, some embodiments provide for other, site-specificmodifications at, or within close proximity to, the identified mutation.Whereas most attenuating mutations produced in biologically-derivedBunyaviridae are single nucleotide changes, other “site specific”mutations can also be incorporated by recombinant techniques intobiologically-derived or recombinant Bunyaviridae. As used herein,site-specific mutations include insertions, substitutions, deletions orrearrangements of from 1 to 3, up to about 5-15 or more alterednucleotides (e.g., altered from a wild-type Bunyaviridae family membersequence, from a sequence of a selected mutant Bunyaviridae strain, orfrom a parent recombinant Bunyaviridae clone subjected to mutagenesis).Such site-specific mutations may be incorporated at, or within theregion of, a selected, biologically-derived point mutation.Alternatively, the mutations can be introduced in various other contextswithin an Bunyaviridae clone, for example at or near a cis-actingregulatory sequence or nucleotide sequence encoding a protein activesite, binding site, immunogenic epitope, etc. Site-specific Bunyaviridaemutants typically retain a desired attenuating phenotype, but mayexhibit substantially altered phenotypic characteristics unrelated toattenuation, e.g., enhanced or broadened immunogenicity, or improvedgrowth. Further examples of desired, site-specific mutants includerecombinant Bunyaviridae designed to incorporate additional, stabilizingnucleotide mutations in a codon specifying an attenuating pointmutation. Where possible, two or more nucleotide substitutions areintroduced at codons that specify attenuating amino acid changes in aparent mutant or recombinant Bunyaviridae clone, yielding abiologically-derived or recombinant Bunyaviridae having geneticresistance to reversion from an attenuated phenotype. In otherembodiments, site-specific nucleotide substitutions, additions,deletions or rearrangements are introduced upstream (N-terminaldirection) or downstream (C-terminal direction), e.g., from 1 to 3, 5-10and up to 15 nucleotides or more 5′ or 3′, relative to a targetednucleotide position, e.g., to construct or ablate an existing cis-actingregulatory element.

In addition to single and multiple point mutations and site-specificmutations, changes to chimeric Bunyaviridae disclosed herein includedeletions, insertions, substitutions or rearrangements of whole genes orgene segments. These mutations may alter small numbers of bases (e.g.,from 15-30 bases, up to 35-50 bases or more), or large blocks ofnucleotides (e.g., 50-100, 100-300, 300-500, 500-1,000 bases) in thedonor or recipient genome, depending upon the nature of the change(i.e., a small number of bases may be changed to insert or ablate animmunogenic epitope or change a small gene segment, whereas largeblock(s) of bases are involved when genes or large gene segments areadded, substituted, deleted or rearranged.

In additional aspects, some embodiments provide for supplementation ofmutations adopted into a chimeric Bunyaviridae clone frombiologically-derived Bunyaviridae, e.g., is mutations, with additionaltypes of mutations involving the same or different genes in a furthermodified chimeric Bunyaviridae clone. Bunyaviridae encode 3 mRNAs and 6proteins including L, G_(N), NS_(M), G_(C), N, and NSs. These proteinscan be selectively altered in terms of expression levels, or can beadded, deleted, substituted or rearranged, in whole or in part, alone orin combination, with other desired modifications, to yield a chimericBunyaviridae exhibiting novel vaccine characteristics.

Thus, in addition to, or in combination with, attenuating mutationsadopted from biologically-derived Bunyaviridae mutants, preferredembodiments also provide a range of additional methods for attenuatingchimeric Bunyaviridae based on recombinant engineering of infectiousBunyaviridae clones. In accordance with this aspect, a variety ofalterations can be produced in an isolated polynucleotide sequenceencoding the chimeric Bunyaviridae genome for incorporation intoinfectious clones. More specifically, to achieve desired structural andphenotypic changes in chimeric Bunyaviridae, the invention allows forintroduction of modifications which delete, substitute, introduce, orrearrange a selected nucleotide or plurality of nucleotides from aparent chimeric genome, as well as mutations which delete, substitute,introduce or rearrange whole gene(s) or gene segment(s), within achimeric Bunyaviridae clone.

Desired modifications of infectious chimeric Bunyaviridae are typicallyselected to specify a desired phenotypic change, e.g., a change in viralgrowth, temperature sensitivity, ability to elicit a host immuneresponse, attenuation, etc. These changes can be brought about either ina donor or recipient genome by, e.g., mutagenesis of a parent LACV cloneto ablate, introduce or rearrange a specific gene(s) or gene region(s)(e.g., a gene segment that encodes a protein structural domain, animmunogenic epitope, binding region, active site, etc.). Genes ofinterest in this regard include all of the genes of the LACV genome: L,G_(N), NS_(M), G_(C), N and NSs, and a variety of genes from otherviruses of the Bunyaviridae family.

Also provided are modifications in a chimeric Bunyaviridae which simplyalter or ablate expression of a selected gene, e.g., by introducing atermination codon within a selected Bunyaviridae coding sequence,changing the position of an Bunyaviridae gene relative to an operablylinked promoter, introducing an upstream start codon to alter rates ofexpression, modifying (e.g., by changing position, altering an existingsequence, or substituting an existing sequence with a heterologoussequence) transcription signals to alter phenotype (e.g., growth,temperature restrictions on transcription, etc.), and various otherdeletions, substitutions, additions and rearrangements that specifyquantitative or qualitative changes in viral replication, transcriptionof selected gene(s), or translation of selected protein(s).

The ability to analyze and incorporate other types of attenuatingmutations into chimeric Bunyaviridae for vaccine development extends toa broad assemblage of targeted changes in Bunyaviridae clones. In someembodiments, a gene deletion is combined in a chimeric Bunyaviridae withone or more additional mutations specifying an attenuated phenotype,e.g., one or more point mutation(s) adopted from a biologically-derived,attenuated Bunyaviridae family member mutant.

In this regard, any Bunyaviridae gene that is not essential for growthcan be ablated or otherwise modified in a chimeric Bunyaviridae to yielddesired effects on virulence, pathogenesis, immunogenicity and otherphenotypic characteristics.

In addition, a variety of other genetic alterations can be produced in aBunyaviridae genome for incorporation into infectious chimericBunyaviridae, alone or together with one or more attenuating pointmutations adopted from a biologically-derived mutant Bunyaviridae.Additional heterologous genes and gene segments (e.g., from differentBunyaviridae genes, different Bunyaviridae strains or types) may beinserted in whole or in part, the order of genes changed, gene overlapremoved, portions of genes removed or substituted, and even entire genesdeleted. Different or additional modifications in the sequence can bemade to facilitate manipulations, such as the insertion of uniquerestriction sites in various intergenic regions or elsewhere.Nontranslated gene sequences can be removed to increase capacity forinserting foreign sequences.

Also provided herein are genetic modifications in a chimericBunyaviridae family member which alter or ablate the expression of aselected gene or gene segment without removing the gene or gene segmentfrom the chimeric Bunyaviridae clone. For example, this can be achievedby introducing a termination codon within a selected coding sequence,changing the position of a gene or introducing an upstream start codonto alter its rate of expression. Other mutations within chimericBunyaviridae involve replacement of the 5′ or 3′ ends of the genome.

In another exemplary embodiment, a sequence surrounding a translationalstart site (preferably including a nucleotide in the −3 position) of aselected Bunyaviridae gene is modified, alone or in combination withintroduction of an upstream start codon, to modulate chimericBunyaviridae gene expression by specifying up- or down-regulation oftranslation.

In more embodiments, chimeric Bunyaviridae are provided in whichexpression of a viral gene, for example the NSs gene, is ablated at thetranslational level without deletion of the gene or of a segmentthereof, by, e.g., introducing two tandem translational terminationcodons into a translational open reading frame (ORF) (e.g., therLACVdeINSs mutation (Blakqori, G. and Weber F. 2005 J Virol79:10420-10428). This yields viable virus in which a selected gene hasbeen silenced at the level of translation, without deleting its gene.These forms of “knock-out” virus will exhibit reduced growth rates andsmall plaque sizes in tissue culture. Thus, the methods and compositionsdescribed herein provide yet additional, novel types of attenuatingmutations which ablate expression of a viral gene that is not one of themajor viral protective antigens. In this context “knockout” virusphenotypes produced without deletion of a gene or gene segment can bealternatively produced by deletion mutagenesis, as described herein, toeffectively preclude correcting mutations that may restore synthesis ofa target protein.

Several other gene “knock-outs” for chimeric Bunyaviridae can be madeusing alternate designs. For example, insertion of translationtermination codons into ORFs, or disruption of the RNA editing sites,offer alternatives to silencing or attenuating the expression ofselected genes. Methods for producing these and other knock-outs arewell known in the art.

Infectious chimeric Bunyaviridae clones can also be engineered accordingto the methods and compositions disclosed herein to enhanceimmunogenicity and induce a level of protection greater than thatprovided by infection with a wild-type Bunyaviridae or a parent chimericBunyaviridae. For example, an immunogenic epitope from a heterologousLACV strain or type, or from a non-LACV source such as a hantavirus, canbe added to a chimeric clone by appropriate nucleotide changes in thepolynucleotide sequence encoding the chimeric genome. Alternatively,chimeric Bunyaviridae can be engineered to add or ablate (e.g., by aminoacid insertion, substitution or deletion) immunogenic epitopesassociated with desirable or undesirable immunological reactions.

Within the methods described herein, additional genes or gene segmentsmay be inserted into the recipient Bunyaviridae genome. These genes maybe under common control with recipient genes, or may be under thecontrol of an independent set of transcription signals. Genes ofinterest include the Bunyaviridae genes identified above, as well asnon-Bunyaviridae genes. Non-Bunyaviridae genes of interest include thoseencoding cytokines (e.g., IL-2 through IL-15, especially IL-2, IL-6 andIL-12, etc.), gamma-interferon, and proteins rich in T-helper cellepitopes. These additional proteins can be expressed either as aseparate protein, or as a chimera engineered from a second copy of oneof the Bunyaviridae proteins. This provides the ability to modify andimprove the immune responses against Bunyaviridae both quantitativelyand qualitatively.

In exemplary embodiments, insertion of foreign genes or gene segments,and in some cases of noncoding nucleotide sequences, within a chimericBunyaviridae genome results in a desired increase in genome lengthcausing yet additional, desired phenotypic effects. Increased genomelength results in attenuation of the resultant Bunyaviridae, dependentin part upon the length of the insert. In addition, the expression ofcertain proteins, e.g., a cytokine, from a non-Bunyaviridae geneinserted into chimeric Bunyaviridae as described herein will result inattenuation of the virus due to the action of the protein. This has beendescribed in the scientific literature for IL-2 expressed in vacciniavirus and is also expected for gamma interferon.

Deletions, insertions, substitutions and other mutations involvingchanges of whole viral genes or gene segments within chimericBunyaviridae yield highly stable vaccines, which are particularlyimportant in the case of immunosuppressed individuals. Many of thesechanges will result in attenuation of resultant vaccine strains, whereasothers will specify different types of desired phenotypic changes. Forexample, certain viral genes are known, which encode proteins thatspecifically interfere with host immunity. Ablation of such genes inchimeric vaccine viruses is expected to reduce virulence andpathogenesis and/or improve immunogenicity.

In addition to the above described modifications to recombinantBunyaviridae, different or additional modifications in Bunyaviridaeclones can be made to facilitate manipulations, such as the insertion ofunique restriction sites in various intergenic regions. Nontranslatedgene sequences can be removed to increase capacity for inserting foreignsequences.

In another aspect of the invention, compositions (e.g., isolatedpolynucleotides and vectors incorporating a chimericBunyaviridae-encoding cDNA) are provided for producing an isolatedinfectious chimeric Bunyaviridae. In some aspects of the invention theDNAs encoding the immunogens described herein are codon-optimized forexpression in a particular host (e.g., codon-optimized for expression inhumans). These codon optimized DNA immunogens can be introduced into amammalian subject (e.g., human) by conventional DNA vaccinationtechniques as described herein including electroporation, gene gun,powder injection, aerosol, and inhalation. Using these compositions andmethods, infectious chimeric Bunyaviridae are generated from a chimericBunyaviridae genome. In related aspects, compositions and methods areprovided for introducing the aforementioned structural and phenotypicchanges into a recombinant chimeric Bunyaviridae to yield infectious,attenuated vaccine viruses.

Introduction of the foregoing defined mutations into an infectious,chimeric Bunyaviridae clone can be achieved by a variety of well-knownmethods. By “infectious clone” is meant cDNA or its product, syntheticor otherwise, which can be transcribed into genomic RNA capable ofserving as template to produce the genome of an infectious virus orsubviral particle. Thus, defined mutations can be introduced byconventional techniques (e.g., site-directed mutagenesis) into a cDNAcopy of the genome. The use of genome cDNA subfragments to assemble acomplete genome cDNA as described herein has the advantage that eachregion can be manipulated separately (smaller cDNAs are easier tomanipulate than large ones) and then readily assembled into a completecDNA. Thus, the complete genome cDNA, or any subfragment thereof, can beused as template for oligonucleotide-directed mutagenesis. This can bethrough the intermediate of a single-stranded phagemid form, such asusing the Muta-Gene® kit of Bio-Rad Laboratories (Richmond, Calif.) or amethod using a double-stranded plasmid directly as template such as theChameleon mutagenesis kit of Stratagene (La Jolla, Calif.), or by thepolymerase chain reaction employing either an oligonucleotide primer ortemplate which contains the mutation(s) of interest. A mutatedsubfragment can then be assembled into the complete genome cDNA. Avariety of other mutagenesis techniques are known and available for usein producing the mutations of interest in the Bunyaviridae genome cDNA.Mutations can vary from single nucleotide changes to replacement oflarge cDNA pieces containing one or more genes or genome regions.

Thus, in one illustrative embodiment mutations are introduced by usingthe Muta-gene phagemid in vitro mutagenesis kit available from Bio-Rad.In brief, cDNA encoding a portion of a Bunyaviridae genome is clonedinto the plasmid pTZ18U, and used to transform CJ236 cells (LifeTechnologies). Phagemid preparations are prepared as recommended by themanufacturer. Oligonucleotides are designed for mutagenesis byintroduction of an altered nucleotide at the desired position of thegenome. The plasmid containing the genetically altered genome fragmentis then amplified and the mutated piece is then reintroduced into thefull-length genome clone.

The ability to introduce defined mutations into infectious Bunyaviridaehas many applications, including the analyses of viral molecular biologyand pathogenesis. For example, the functions of the Bunyaviridaeproteins, including the L, G_(N), NS_(M), G_(C), N and NSs proteins, canbe investigated and manipulated by introducing mutations which ablate orreduce their level of expression, or which yield mutant protein.

By “recombinant Bunyaviridae” is meant a Bunyaviridae orBunyaviridae-like viral or subviral particle derived directly orindirectly from a recombinant expression system or propagated from virusor subviral particles produced therefrom. The recombinant expressionsystem will employ a recombinant expression vector which comprises anoperably linked transcriptional unit comprising an assembly of at leasta genetic element or elements having a regulatory role in Bunyaviridaegene expression, for example, a promoter, a structural or codingsequence which is transcribed into Bunyaviridae RNA, and appropriatetranscription initiation and termination sequences.

To produce infectious Bunyaviridae from cDNA-expressed genome, thegenome is coexpressed with those Bunyaviridae proteins necessary to (i)produce nucleocapsids capable of RNA replication, and (ii) renderprogeny nucleocapsids competent for both RNA replication andtranscription. Transcription by the genome nucleocapsids provide theother Bunyaviridae proteins and initiates a productive infection.Alternatively, additional Bunyaviridae proteins needed for a productiveinfection can be supplied by coexpression.

In certain embodiments, complementing sequences encoding proteinsnecessary to generate transcribing, replicating Bunyaviridaenucleocapsids are provided by one or more helper viruses. Such helperviruses can be wild-type or mutant. Preferably, the helper virus can bedistinguished phenotypically from the virus encoded by the BunyaviridaecDNA. For example, it is desirable to provide monoclonal antibodieswhich react immunologically with the helper virus but not the virusencoded by the Bunyaviridae cDNA. Such antibodies can be neutralizingantibodies. In some embodiments, the antibodies can be used in affinitychromatography to separate the helper virus from the recombinant virus.To aid the procurement of such antibodies, mutations can be introducedinto the Bunyaviridae cDNA to provide antigenic diversity from thehelper virus.

Alternative means to construct cDNA encoding a Bunyaviridae genomeinclude reverse transcription-PCR using PCR conditions to reduce thenumber of subunit cDNA components to as few pieces as possible. In otherembodiments different promoters can be used (e.g., T7, T3, SP6).Different DNA vectors (e.g., cosmids) can be used for propagation tobetter accommodate large size genome.

To select candidate chimeric vaccine viruses, the criteria of viability,attenuation and immunogenicity are determined according to well-knownmethods. Viruses which will be most desired in vaccines and immunogeniccompositions as described herein preferably maintain viability, have astable attenuation phenotype, exhibit replication in an immunized host(albeit at lower levels), and effectively elicit production of an immuneresponse in a vaccinee sufficient to confer protection against seriousdisease caused by subsequent infection from wild-type virus.

Chimeric Bunyaviridae, which have been attenuated as described hereincan be tested in various well known and generally accepted in vitro andin vivo models to confirm adequate attenuation, resistance to phenotypicreversion, and immunogenicity for vaccine use. In in vitro assays, themodified virus (e.g., a multiply attenuated, biologically-derived orrecombinant Bunyaviridae) is tested for temperature sensitivity of virusreplication, i.e., is phenotype, and for the small plaque phenotype.Modified viruses are further tested in animal models of Bunyaviridaeinfection.

In accordance with the foregoing description, some embodiments alsoconcern isolated, infectious chimeric Bunyaviridae compositions forvaccine or immunogenic composition use. The attenuated chimeric viruswhich is a component of a vaccine is in an isolated and typicallypurified form. By “isolated” is meant to refer to virus which is inother than a native environment of a wild-type virus. More generally,“isolated” is meant to include the attenuated virus as a component of acell culture or other artificial medium. For example, attenuatedBunyaviridae of the invention may be produced by an infected cellculture, separated from the cell culture and added to a stabilizer whichcontains other non-naturally occurring Bunyaviridae family members, suchas those that are selected to be attenuated by means of resistance toneutralizing monoclonal antibodies.

Chimeric Bunyaviridae vaccines and immunogenic compositions contain asan active ingredient an immunogenically effective amount of Bunyaviridaeproduced as described herein. Biologically derived or recombinantBunyaviridae can be used directly in vaccine formulations, orlyophilized. Lyophilized virus will typically be maintained at about 4°C. When ready for use the lyophilized virus is reconstituted in astabilizing solution, e.g., saline, with or without adjuvant, as furtherdescribed below. The biologically-derived or recombinantly modifiedvirus may be introduced into a host with a physiologically acceptablecarrier and/or adjuvant. Useful carriers are well known in the art, andinclude, e.g., water, buffered water, 0.4% saline, 0.3% glycine,sucrose, hyaluronic acid and the like. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration, as mentioned above. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, and the like. Acceptable adjuvants include incomplete Freund'sadjuvant, aluminum phosphate, aluminum hydroxide, or alum, which arematerials well known in the art. Preferred adjuvants also includeStimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Farmingham, Mass.),MPL™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research,Inc., Hamilton, Mont.), and interleukin-12 (Genetics Institute,Cambridge, Mass.).

Upon immunization with a chimeric Bunyaviridae vaccine or immunogeniccomposition as described herein, via parenteral, oral, topical or otherroute, the immune system of the host responds to the vaccine byproducing antibodies specific for one or more Bunyaviridae proteins.Preferably, in the methods described herein, the host immune response(e.g., T cell recall or antibody titer) is measured. Such measurementscan be made by conventional assays. Clinical evaluation and observationof an improvement in condition can also be a measurement of immuneresponse in some contexts. As a result of the vaccination/inoculationthe host becomes at least partially or completely immune to Bunyaviridaeinfection, or resistant to developing moderate or severe Bunyaviridaedisease.

Chimeric Bunyaviridae vaccines and/or immunogenic compositions may alsocomprise attenuated chimeric virus that elicits an immune responseagainst a single Bunyaviridae strain or antigenic subgroup, or againstmultiple Bunyaviridae strains or subgroups. In this context, thechimeric Bunyaviridae can elicit a monospecific immune response or apolyspecific immune response against multiple Bunyaviridae strains orsubgroups. Alternatively, chimeric Bunyaviridae having differentimmunogenic characteristics can be combined in a vaccine mixture oradministered separately in a coordinated treatment protocol to elicitmore effective protection against one Bunyaviridae strain, or againstmultiple Bunyaviridae strains or subgroups.

The host to which the vaccine/immunogenic composition is administered orprovided can be any mammal susceptible to infection by a particularvirus of the Bunyaviridae family or a closely related virus and capableof generating a protective immune response to antigens of thevaccinizing virus. Thus, suitable hosts include humans, non-humanprimates, bovine, equine, swine, ovine, rodents, etc. Accordingly, theinvention provides methods for creating vaccines for a variety of humanand veterinary uses.

The vaccine and immunogenic compositions containing the attenuatedchimeric Bunyaviridae as described herein are administered or providedto an individual susceptible to or otherwise at risk of Bunyaviridaeinfection in an “immunogenically effective dose” which is sufficient toinduce or enhance the individual's immune response capabilities againstBunyaviridae. In the case of human subjects, the attenuated virus isadministered or provided according to well established human vaccineprotocols.

In all subjects, the precise amount of chimeric Bunyaviridae vaccineadministered or provided and the timing and repetition of inoculationwill be determined based on the patient's state of health and weight,the mode of administration, the nature of the formulation, etc. Dosageswill generally range from about 10² to about 10⁶ plaque forming units(PFU) or more of virus per individual, more commonly from about 10³ to10⁵ PFU virus per individual. In any event, the vaccine formulationspreferably provide a quantity of attenuated virus as described hereinsufficient to effectively stimulate or induce an anti-Bunyaviridaeimmune response, e.g., as can be determined by complement fixation,plaque neutralization, and/or enzyme-linked immunosorbent assay, amongother methods. In this regard, individuals are also monitored for signsand symptoms of illness. As with administration to chimpanzees, theattenuated virus of the vaccine grows in vaccinees at levelsapproximately 10-fold or more lower than wild-type virus, orapproximately 10-fold or more lower when compared to levels ofincompletely attenuated virus.

In neonates and infants, multiple administration may be required toelicit sufficient levels of immunity. Administration should begin withinthe first month of life, and at intervals throughout childhood, such asat two months, six months, one year and two years, as necessary tomaintain sufficient levels of protection against native (wild-type)Bunyaviridae infection. Similarly, adults who are particularlysusceptible to repeated or serious Bunyaviridae infection, such as, forexample, health care workers, day care workers, family members of youngchildren, the elderly, individuals with compromised cardiopulmonaryfunction, may require multiple immunizations to establish and/ormaintain protective immune responses. Levels of induced immunity can bemonitored by measuring amounts of neutralizing secretory and serumantibodies, and dosages adjusted or vaccinations repeated as necessaryto maintain desired levels of protection. Further, different vaccineviruses may be indicated for administration to different recipientgroups. For example, an engineered chimeric Bunyaviridae strainexpressing a cytokine or an additional protein rich in T cell epitopesmay be particularly advantageous for adults rather than for infants.Bunyaviridae vaccines produced in accordance with the present inventioncan be combined with viruses expressing antigens of another subgroup orstrain of Bunyaviridae to achieve protection against multipleBunyaviridae subgroups or strains. Alternatively, the vaccine virus mayincorporate protective epitopes of multiple Bunyaviridae strains orsubgroups engineered into one Bunyaviridae clone as described herein.

Typically when different vaccine viruses are used they will beadministered in an admixture simultaneously, but they may also beadministered separately. Immunization with one strain may protectagainst different strains of the same or different subgroup.

The level of attenuation of chimeric vaccine virus may be determined by,for example, quantifying the amount of virus present in an immunizedhost and comparing the amount to that produced by wild-type Bunyaviridaeor other attenuated Bunyaviridae which have been evaluated as candidatevaccine strains. For example, the attenuated chimeric virus of theinvention will have a greater degree of restriction of replication in ahighly susceptible host, such as a chimpanzee, compared to the levels ofreplication of wild-type virus, e.g., 10- to 1000-fold less.

Genome Sequence Analysis of La Crosse Virus and In Vitro and In VivoPhenotypes

LACV genomes were sequenced for two reasons. First, we wanted todetermine the genetic diversity of LACV isolated in different regions ofthe United States at different times, and second, we sought to define acomplete genomic sequence that is associated with the wild typephenotype of virulence in mice by both peripheral and intracerebralroutes of inoculation. The sequence of only two complete LACV genomeswas previously reported, one human isolate (LACV/human/1978, GenBankaccession numbers NC_004108-NC_004110) and one mosquito isolate (strain77, LACV/mosquito/1977, GenBank accession numbers DQ196118-DQ19620)(Table B). We sequenced two additional isolates, including the originalLa Crosse, Wis. virus (LACV/human/1960) and the LACV/mosquito/1978 virus(GenBank accession numbers EF485036-EF485038) (Table B), andre-sequenced LACV/human/1978 after an additional passage in tissueculture to confirm its identity prior to further biologicalcharacterization. See Virology Journal 4:41(2007), herein expresslyincorporated by reference in its entirety). In addition,LACV/human/1960, LACV/mosquito/1978, and LACV/human/1978 parentalwild-type viruses were biologically cloned to generate a geneticallyhomogeneous viral preparation, and the full sequence of these clonedpreparations was also determined. Thus, we have generated full-lengthsequence for 3 pairs of cloned and uncloned LACV strains. ForLACV/human/1978, the newly derived sequence (EF485033-35) was used forall subsequent comparisons since several differences with the formersequence were identified. An examination of the virulence phenotype ofthese three parental and cloned viruses in mice should define one ormore full-length sequences that have a wild type phenotype.

TABLE B Passage history and geographic location of isolation/infectionof the LACV isolates for which complete genomic sequences are available.GenBank Accession Virus Location Passage history^(a) numberLACV/human/1960 Minnesota C6/36 2 EF485030-EF48532 LACV/human/1960-cloneMinnesota C6/36 2, Vero 4 NA^(b) LACV/mosquito/1978 North Carolina Mousebrain 1, Vero 3 EF485036-EF48538 LACV/mosquito/1978-clone North CarolinaMouse brain 1, Vero 7 NA LACV/human/1978 Wisconsin Mouse brain 1, BHK 2,Vero 1 EF485033-EF48535 LACV/human/1978-clone Wisconsin Mouse brain 1,BHK 2, Vero 5 NA LACV/mosquito/1977 Wisconsin UnknownDQ196118-DQ19620^(c) LACV/human/1978 Wisconsin Mouse brain 1, BHK 2 NC004108-NC 004110^(d) ^(a)Cell/tissue type followed by number ofpassages. ^(b)Sequence not submitted. Genetic comparisons with unclonedparental wildtype stocks found in Table 3. ^(c)Previous submission byCheng et al. 2005. ^(d)Previous submission by Hughes et al. 2002.Sequence from a derivative of this virus (one additional passage in Verocells) was generated for this study (EF485033-EF48535) and used forsubsequent comparisons since several differences with this sequence wereidentified.

A comparison of the complete genomic nucleotide sequences from lowpassage LACV isolates of either human or mosquito origin isolated overan 18 year period of time from two geographically different regions ofthe United States (Table C) indicated little sequence divergence. The S,M, and L genome segments for each virus were 984, 4526, and 6980nucleotides in length, respectively. The nucleotide length of thesegments and the encoded open reading frames for each of the isolatesare identical. The S, M, and L segments from each virus isolate share ahigh nucleotide sequence identity ranging from 97.9-100%, 95.7-99.8%,and 95.7-99.4% respectively (Table C). The N, NSs, M polyprotein, and Lprotein open reading frames are 235, 92, 1441, and 2263 amino acidcodons in length, respectively. The percent identity for encodedproteins is also highly conserved among the isolates with 99.6-100%,98.9-100%, 97.8-99.6%, and 99.2-99.7% identity for the N, NSs, Mpolyprotein, and L proteins, respectively (Table C).

TABLE C Nucleotide and amino acid identity (%) of the LACV genomicsegments and their predicted protein products. Mos- Mos- Human/ Human/quito/ quito/ 1960 1978 1978 1977 S segment/N protein Human/1960Nucleotide — 99.6 100   100 Amino Human/1978 99.8 — 99.6 99.6 AcidMosquito/1978 98.1 97.9 — 100 Mosquito/1977 100   99.8 98.1 — Ssegment/NSs protein Human/1960 Nucleotide — 98.9 100   100 AminoHuman/1978 99.8 — 98.9 98.9 Acid Mosquito/1978 98.1 97.9 — 100Mosquito/1977 100   99.8 98.1 — M segment/M Polyprotein Human/1960Nucleotide — 99.4 97.8 99.6 Amino Human/1978 99.6 — 97.8 99.4 AcidMosquito/1978 95.8 95.8 — 97.7 Mosquito/1977 99.8 99.5 98.1 — Lsegment/L Protein Human/1960 Nucleotide — 99.7 99.2 99.5 AminoHuman/1978 99.4 — 99.2 99.5 Acid Mosquito/1978 95.9 95.7 — 99.5Mosquito/1977 97.1 96.9 96.0 —

Although the process of biological cloning resulted in an additionalfour passages in Vero cells, these passages had a minimal effect ongenetic stability. A maximum of four nucleotide changes were observedbetween a wild type parental virus stock and its biologically clonedderivative, and no more than one amino acid change was observed in anycloned virus (Table D). This level of sequence divergence betweenparental and cloned virus is much less than that between LACV isolates(>500 nucleotides differences for human/1978 compared to mosquito/1978),clearly identifying each isolate as a separate strain.

TABLE D Nucleotide differences between wild type parental andbiologically cloned virus. Nucleotide Substitution in indicated LACVsegment^(a) LACV Virus S M L human/1960-clone A525T A503G^(b) A1837GC2221T human/1978-clone No changes^(c) T391C No changes A1636GA1929G^(b) mosquito/1978-clone A719G^(b) No changes A31G G33A^(a)Parental nucleotide on left, nucleotide substitution in the clonedvirus on right. ^(b)Indicates a nucleotide substitution resulting in anamino acid substitution. ^(c)Parental and cloned virus have identicalsequences.

The sequences of two LACV isolates from humans were compared with twoisolates from mosquitoes to identify amino acids that are shared byLACVs of human origin but that differ from LACVs of mosquito origin.Such sequence differences are referred to as host-specific sequencesubstitutions. Five such host-specific amino acid substitutions wereidentified, and all were located in the L protein (Table E). Fournucleotide substitutions in the non-coding region (NCR) of the L segmentat nucleotides 31, 6876, 6877, and 6921 also appear to be host-specific(FIGS. 1 and 2).

TABLE E Host specific amino acid differences are located in the RNApolymerase (L). Amino acid residue Amino acid position Human Mosquito129 V I 484 K R 1040 E G 1713 T A 1906 A S Genetic comparisons used allwild-type parental sequences generated for this paper and publishedsequences of mosquito strain LACV-77 (DQ196118-DQ19620). RNA polymeraseamino acid 922 was R or G in the human isolates and K in the twomosquito isolates.

The 3′ and 5′ genome ends of the LACV genomes were also highlyconserved. The first 11 and last 11 nucleotides were identical for eachsegment end (FIGS. 1 and 2). Each 3′ NCR was identical for the S (nt1-81) and only one nucleotide differed from the consensus in the M (nt1-61) segments. The 3′ NCR of L (nt 1-61) from LACV/mosquito/1978differed from the consensus by 2 nucleotides (FIG. 1). The 5′ NCR ofLACV/mosquito/1978 differed from the S, M, and L consensus by 8, 3, and1 nucleotides, respectively. For the L segment, a clear consensussequence was not identified at position 31 of the 3′ end and atnucleotide positions 6876, 6877, 6888, and 6921 in the 5′ NCR. Betweenthe two human isolates, one nucleotide difference in the NCR wasidentified at position 6888 of the 5′ NCR of L (FIG. 2).

In Vitro Growth Kinetics

Comparison of in vitro growth of LACV/human/1960, LACV/mosquito/1978 andLACV/human/1978 viruses was performed in Vero cells and C6/36 cells(FIG. 3). All viruses replicated to high titers in both cell types.LACV/human/1960 replicated more quickly in C6/36 cells, possibly as aresult of originally being isolated in these cells. Growth kinetics inVero cells for all three viruses were nearly identical reachingapproximately 10⁷ PFU/ml in 24 hours (FIG. 3A). Each of the threeviruses replicated efficiently in C6/36 cells, but, in contrast to therapid development of cytopathic effects (CPE) in Vero cells, infectionof C6/36 cells was not cytopathic over the seven day period. CPEassociated with LACV infection of Vero cells consisted of cell roundingand detachment from the flask with 80% of the monolayer destroyed by 3days post-infection (FIG. 3B).

LACV Clinical Disease in Mice: LD₅₀

For LACV, clinical disease in mice is described by two characteristics:neurovirulence and neuroinvasiveness. Neurovirulence, defined as theextent to which a virus can infect tissues of the brain, is determinedby direct inoculation of the virus into the brain followed byobservation of the clinical outcome, in this case, mortality.Neuroinvasiveness is the ability of a virus introduced outside of thecentral nervous system (via intraperitoneal inoculation in this case) togain access and infect the brain, which is also observed as mortality.All six LACV isolates were neurovirulent in Swiss Webster miceregardless of previous passage in mouse brains. Clinical disease in miceincluded lethargy, tremors, seizures, and limb paralysis, although therewas no consistent sequence to the progression of disease. In suckling orweanling mice, the LD₅₀ values ranged from −0.50 to 1.50 log₁₀ PFU(Table F). Thus, each of the three genome sequences of the parental andcloned viruses is a sequence of a fully neurovirulent virus. Of the sixLACV isolates tested, five were neuroinvasive for mice of both ageswhereas the LACV/human/1960-clone did not induce clinical disease inweanling mice even after inoculation of 10⁶ PFU (Table F). It wasdetermined that this virus has a nucleotide substitution (A503G)resulting in a single amino acid change at position 148(Threonine-Alanine) in the G_(N) (formerly G2) attachment glycoproteinand three silent nucleotide substitutions (Table D). This suggests thatthe alanine residue at position 148 attenuates neuroinvasiveness. Asshown in FIGS. 4-6, Threonine is well-conserved at this amino acidposition among the other virus members of the CEV serogroup.

TABLE F La Crosse neurovirulence and neuroinvasiveness afterintracerebral (IC) or intraperitoneal (IP) inoculation of Swiss Webstermice. Neurovirulence (IC) Neuroinvasiveness (IP) (LD₅₀ log₁₀ PFU) (LD₅₀log₁₀ PFU) Suckling Weanling Suckling Weanling Virus mice^(a) mice^(b)mice mice LACV/human/1960 1.35 1.30 2.17 2.56 LACV/human/1960- 1.37−0.25 1.76 >6.0 clone^(c) LACV/human/1978 0.37 −0.50 0.57 1.75LACV/human1978- 0.42 −0.15 0.83 1.25 clone LACV/mosquito/1978 1.19 1.131.08 1.84 LACV/mosquito/1978- 1.36 1.50 1.29 2.40 clone ^(a)Sucklingmice are 2-3 days old. ^(b)Weanling mice are 21-23 days old.^(c)Biologically cloned viruses were obtained by terminally dilutingwild type parental virus three times, then amplified by an additionalpassage in tissue culture.

As an initial step in vaccine development, we defined a panel of LACVgenomic sequences that is associated with wild type in vitro and in vivophenotypes. This was done by examining the phenotypic properties ofparental and biologically cloned derivatives of three LACV viruses. Forour purpose, a LACV was defined as exhibiting a wild type phenotype ifit was fully replication competent in insect and mammalian cells and wasable to cause encephalitic disease in suckling and weanling mice by aperipheral and intracerebral route of inoculation. Although LACV doesnot appear virulent in either mosquitoes or in its amplifying hosts innature, it is clearly virulent in humans and mice resulting in severecentral nervous system (CNS) infections in both species. Therefore, thisvirulence for CNS of mice is the phenotype that we envision as beingmodified as a surrogate phenotype for the development of attenuatedvaccines for humans. Five of the six LACV isolates studied, threeparental and two cloned viruses, had the wild type virulence phenotypein vivo. These defined wild type sequences can now be used as a baselinefor the identification of mutations that attenuate LACV for the CNS. Alive, attenuated LACV virus vaccine could reduce the occurrence of LACVencephalitis in the U.S., and could also be useful as a geneticbackground for the creation of chimeric vaccines against other pathogensin the Bunyaviridae family as has been successfully done for theflaviviruses and paramyxoviruses (Buchholz U J et al. 2000 J Virol74:1187-1199; Blaney J E, Jr. et al. 2006 Viral Immunol 19:10-32).

In the present study, the complete genomic sequence of two wild typeLACV isolates was determined, and one previously determined sequence wasconfirmed with some minor clarifications of the published sequences.These three LACV sequences, along with a previously determined sequence,were compared to examine the extent of genetic diversity of the LACVgenome. Although these four viruses originated in distinct geographiclocations and were isolated from either humans or mosquitoes over aperiod of 18 years, the viruses exhibited a remarkable level of geneticrelatedness independent of passage history, location of isolation, orhost. The N open reading frame of the S segment was the most conservedprotein sequence (>98% identity) among the isolates, followed by that ofthe RNA polymerase L, NSs, and M polyprotein (>95% identity). It ispossible that ecological factors, such as the need to replicateefficiently in both mammalian and insect hosts, have selected for agenotype that has obtained maximum fitness in both hosts. In such amodel, variants that arise by genetic drift may be quickly selectedagainst by either host. Although recovery of LACV from humans is unusualand isolates are rare, a greater number of isolates from both hostsmight be examined for further sequence analysis.

Since we had complete sequences for two viruses isolated from mosquitoesand two from humans, it was possible to search for host specificsequences that distinguish between LACV isolates obtained from the twospecies. Five amino acid substitutions were found in the RNA polymeraseand may define the host-specific genetic differences. In addition, fournucleotide differences in the NCR of the L segment also appear to behost-specific. Such host-specific differences were not identified in theS or M segments. None of the differences is located in the conservedBunyaviridae L protein motifs A-D (Roberts A et al. 1995 Virology206:742-745). Since the human isolates would only be obtained fromsymptomatic cases, it is possible that the L segment of the LACV mightbe a determinant of virulence in humans and that only those LACVs with aspecific L segment sequence are isolated from humans with disease. Sincethere were nine host-specific differences between the human and mosquitoisolates, it is unlikely, although not impossible, that nine sharedchanges would have co-developed during the replication of a LACV in twodifferent humans following infection with the mosquito genotype. Rather,it is likely that there are subsets of LACV strains in nature, only someof which might be capable of causing severe disease in humans. Sincevirus with mosquito- or human-specific L segment sequences did notdiffer in virulence in mice, this suggestion of an association of asequence with human disease is offered with great caution. As additionalvirus isolates from humans become available for sequence analysis, itwill be important to monitor these specific amino acids and nucleotidesfor their association with human disease. In addition, it will beinteresting to determine if viruses with the human host specificsequences can be directly isolated from mosquitoes.

As a first step towards vaccine development, we sought to establish areproducible murine model of LACV infection suitable for pathogenesisand vaccine safety/efficacy studies. In humans, disease incidence is agedependent with the majority of cases in children under 15 years old(Woodruff B A et al. 1992 Am J Epidemiol 136:320-327). Previous studiesin BALB/C mice using LACV/human/1960, passaged nine times in sucklingmouse brain and two times in BHK cells, resulted in an age-specificdecrease in neuroinvasiveness most notable at 3 weeks of age (Janssen Ret al. 1984 Lab Invest 50:447-455). In our Swiss Webster mouse model,the LD₅₀ values were similar for both age groups, with the exception ofthe biologically cloned LACV/human/1960. This virus was neuroinvasive insuckling mice but not in weanling mice presumably due to a mutation inthe G_(N) glycoprotein. Although the G_(N) glycoprotein is believed toplay a role in binding of LACV virions to mosquito midgets, it may alsohave a role in the development of CNS disease (Ludwig G V et al. 1991Virology 181:564-571). The use of 3-week-old weanling mice wasadvantageous because they are more mobile than suckling mice allowingfor a more detailed observation of clinical disease manifestations.Following inoculation of 5-week-old Swiss Webster mice with 10⁵ PFU ofLACV/mosquito/1978 (a dose 100% lethal for 3-week-old mice) only 50% (3of 6) became ill, suggesting that Swiss Webster mice will also be usefulin understanding age-dependent neuroinvasiveness of LACV.

Taken together, these results have implications for our future vaccinedevelopment efforts. First, LACV is genetically stable over time anddistance, suggesting that a vaccine based on any of these virus isolatesshould induce a protective immune response against most, if not all,circulating LACV strains. Second, we have identified a mutation in theG_(N) glycoprotein that appears to be associated with decreasedneuroinvasiveness, yet does not affect virus replication in tissueculture. Clearly, our current in vivo testing allows for theidentification of mutations effecting neuroinvasiveness. Third, we haveidentified a convenient mouse model that will allow us to screennumerous mutant viruses for attenuated neuroinvasiveness/neurovirulenceand allow us to continue to evaluate the pathogenesis of LACV infectionand disease.

Example 1

Several experiments were performed to identify mutations in wild-type LaCrosse virus (LACV) that impact various aspects of the virus. Theseexperiments were designed to identify mutations that attenuate the virusin a mammalian subject (e.g., mouse) and, more specifically, to identifymutations that were able to decrease LACV-mediated neurovirulence and/orneuroinvasiveness.

A conventional reverse genetic system for LACV was used to recover virusfrom cDNA clones, which allowed the introduction of mutations into theLACV genome and the rapid evaluation of the attenuated phenotype of thenewly created mutant virus. (See Blakqori et al., J. Virology 79:16(10420-10428) 2005), herein expressly incorporated by reference in itsentirety). The reverse genetics system was used to introduce mutationsinto wild-type LACV (LACV/mosquito/1978, isolated in North Carolina,GenBank accession numbers EF485036-EF485038 and in LACV/human/1960).This analysis revealed one particular mutation (T148A), which attenuateswild-type LACV in mice.

Other approaches to identify additional attenuating mutations wereinvestigated. By one method, a plaque variant technique was employed.Viruses with altered plaque morphologies were isolated fromtissue-culture grown wild-type LACV. Several isolates displayed adecreased level of neuroinvasiveness in mice and the genomes of theseviruses were sequenced (Table G). Mutation P43L (NSs) from the smallplaque isolate and mutation G577E (M) from the large plaque isolate areof particular interest.

TABLE G Level of neuroinvasiveness and amino acids differences amongplaque mutants of LACV/human/1978. Neuro- Amino acid residue at theinvasiveness indicated positions^(a) Virus LD₅₀ in mice NS_(S) Mpolyprotein isolate (log₁₀pfu/mL) N 43 148 577 623 751 L “Small” >4.00 —L T G E N — “Turbid” 1.75 — P T R E N — “Large” >4.00 — P T E E N —Human/ 1.75^(b) — P T G E X — 1978 ^(a)Amino acid number. Unique aminoacids shown in bold. ^(b)Previously determined LD₅₀ value.

By another approach, a technique utilizing 5-fluorouracil (5-FU)mutagenesis was employed. LACV was grown in the presence of the mutagen5-FU and individual viruses were cloned from the mutagenized stock, andscreened for a number of in vitro attenuation phenotypes, including,decreased replication in SH-SY5Y cells (human neuroblastoma), decreasedreplication in LN-18 cells (human glioblastoma cells), and decreasedreplication in C6/36 cells (Aedes mosquito cells) (Table H). Severalvirus isolates displayed a decreased level of neuroinvasiveness in miceand the genomes of these viruses (1C9 and 3E7) were sequenced (Table I).

TABLE H Attenuation phenotypes of selected 5-FU mutant LACV/Human/1960c1Mean virus titer (log₁₀pfu/mL on indicated cell type Neuro- Neuro- SH-LN- virulence invasiveness Vero Y5Y 18 C6/36 e^(a) s^(b) (35° (35° (35°(32° LD₅₀ in mice LD₅₀ in mice Virus C.) C.) C.) C.) (log₁₀pfu/mL)(log₁₀pfu/mL) 1C9 6.7 <0.7 <0.7 5.0 >4.0 >5.0 3E7 6.5 6.2 <0.7<0.7 >4.0 >5.0 wt 7.9 6.9 7.1 7.5 1.30 >6.0 ^(a)Groups of 6 mice eachreceived 10-fold serial dilutions of virus by intracerebral inoculation(0.01 mL) ^(b)Groups of 6 mice each received 10-fold serial dilutions ofvirus by intraperitoneal inoculation (0.1 mL)

TABLE I Amino acids differences among 5-FU mutants of LACV/human/1960c1Amino acid residue at the indicated position in each genome segment^(a)N M^(b) L Virus 110 85 373 588 611 656 131 213 655 1252 1355 1413 18011C9 N A I R E A M G V Y H N E 3E7 S V T M K V I S M C Y D K Wt N V I R EA I S M Y Y D K ^(a)Amino Acid number. ^(b)Mutant and wtLACV/human/1960c1 all contain the T148A mutation in M segment.

Accordingly, it is contemplated that an immunogenic compositioncomprising a peptide that comprises, consists of, or consistsessentially of the T148A mutation or a nucleic acid encoding saidpeptide, which can also be combined with one or more other peptides thatcomprise, consist of, or consist essentially of any one or more othermutations of LACV as described herein, can be manufactured and used toinduce an immune response to LACV (e.g., increase in antibody titer toan LACV antigen) and for the treatment, prevention, or amelioration ofLACV infection or a symptom thereof.

Example 2 Cells and Viruses

C6/36 cells (Aedes albopictus mosquito larvae) were maintained inEarle's MEM supplemented with 10% fetal bovine serum (HyClone), 2 mML-glutamine (Invitrogen), and 1 mM non-essential amino acids. Vero cells(African green monkey kidney) were maintained in OptiPRO™SFM medium(Invitrogen) supplemented with 4 mM L-glutamine (Invitrogen).

LACV/human/1960 was isolated from post-mortem brain tissue collectedfrom a Minnesota patient hospitalized in La Crosse, Wis. and passagedtwo times in C6/36 cells. LACV/mosquito/1978 was isolated frommosquitoes collected in North Carolina and passaged once in mouse brainand three times in Vero cells. LACV/human/1978 was isolated frompost-mortem brain tissue collected in Wisconsin and passaged once inmouse brain, twice in BHK-21 cells, and once in Vero cells (Table B).

Isolation of Biologically Cloned Viruses

Biological clones were generated by terminal dilution in Vero cellcultures. Virus stocks were serially diluted in 2-fold increments andinoculated onto 90% confluent monolayers of Vero cells in 96-well platesusing eight wells per dilution. After five days of incubation, cellculture fluid was removed to a holding plate, and the cell monolayerswere fixed and stained for 10 minutes with crystal violet solution (1%crystal violet in equal volumes of ethanol and methanol). The virus wasselected as a clonal derivative when only 1 or 2 of the 8 wells in asingle row was positive for LACV CPE. Each virus was terminally dilutedthree times (sequentially), amplified in Vero cell culture, andsubjected to genome sequence analysis.

Virus Titrations

Vero cells in 24-well plates were infected in duplicate with ten-foldserial dilutions of LACV and overlayed with OptiMEM (Invitrogen)supplemented with 1% methylcellulose, 5% FBS, 2.5 μg/ml amphotericin B,and 20 μg/ml ciprofloxicin. Five days after infection the overlay wasremoved and cells were washed twice with PBS. The cells were fixed andstained for 10 minutes with crystal violet solution, viral plaques wereidentified by characteristic CPE, and titers are expressed as loginPFU/ml.

Sequence Analysis of Viral Genomes

Viral RNA was isolated using either QIAamp Viral RNA kit (Qiagen) or EZ1Viral RNA mini kit (Qiagen). Reverse transcription (RT) was performedusing random hexamer primers and SuperScript™ First-Strand SynthesisSystem for RT-PCR (Invitrogen). Overlapping PCR fragments were generatedusing LACV specific primers and Advantage cDNA polymerase reaction kit(BD Biosciences Clontech). PCR fragments of up to 2000 bp were purifiedand both strands directly sequenced using viral specific primers inBigDye-terminator cycle sequencing reactions analyzed on an ABI3730genetic analyzer (Applied Biosystems). Sequence fragments were assembledinto a consensus sequence using AutoAssembler 2.1 software (AppliedBiosystems).

To sequence the 5′ and 3′ genome ends, viral RNA was isolated usingQIAamp Viral RNA kit (Qiagen) from virus infected cells at 24-48 hourspost infection for the 3′ non-coding region (NCR) or from cell culturesupernatant fluid for the 5′ NCR. Viral RNA was reverse transcribedusing Reverse Transcriptor (Roche) at 55° C. with random hexamer primersfor the 3′ NCR or at 60-70° C. with genome specific primers thatenhanced reverse transcription though RNA secondary structures. cDNA waspurified with High Pure (Roche) and a poly-A tail was added to the 3′end of the cDNA using 5′/3′ RACE Kit, Second Generation (Roche). Genomeends were then amplified using virus and poly-A specific primers.Purified PCR fragments were sequenced as described above.

In Vitro Growth Kinetics

LACV/human/1960, LACV/mosquito/1978, and LACV/human/1978 were used toinfect 95% confluent monolayers of C6/36 or Vero cells at a multiplicityof infection of 0.01 and incubated for one hour to allow attachment.Infected monolayers were washed twice with sterile PBS and overlaid withmedium. Tissue culture fluid (0.5 ml) was collected every 24 hours afterinfection, mixed 1:10 with 10×SPG buffer (final concentration 218 mMsucrose, 6 mM L-glutamic acid, 3.8 mM dibasic potassium phosphate, pH7.2), and frozen. Daily samples were titrated as described above. Cellmonolayers were photographed on day 0, 1, 2, 3, and 4 forLACV/human/1960 infected, or non-infected Vero cells.

LA CV Clinical Disease in Mice

The lethal doseso (LD₅₀) of LACV virus was evaluated in Swiss Webstersuckling and weanling mice (Taconic Farms, Germantown, N.Y.). All animalexperiments were carried out in accordance with the regulations andguidelines of the National Institutes of Health. Litters of 3 day-oldsuckling mice (n>8/dose) were inoculated with serial dilutions of wildtype or biologically-cloned LACV in a volume of 101l intracerebrally(IC) or 100 μl intraperitoneally (IP). The experiment was repeated with3 week-old weanling mice (n=6/dose), however, the older mice wereanesthetized with isofluorane prior to IC inoculation. All mice werecarefully observed twice daily for clinical disease including tremorsand limb paralysis. Because clinically moribund mice were humanelyeuthanized before succumbing to infection, moribundity served as asurrogate for the determination of lethality.

APPENDIX 1Large genomic segment-nucleotide sequence alignment of LACV/human/1978(Hughes et al. 2002) and LACV/human/1978 (this study) NC_004108AGTAGTGTACCCCTATCTACAAAACTTACAGAAAATTCAGTCATATCACAATATATGCAT   60EF485035 AGTAGTGTACTCCTATCTACAAAACTTACAGAAAATTCAGTCATATCACAATATATGCAT  60 ********** *************************************************NC_004108 AATGGACTATCAAGAGTATCAACAATTCTTGGCTAGGATTAATACTGCAAGGGATGCATG 120 EF485035AATGGACTATCAAGAGTATCAACAATTCTTGGCTAGGATTAATACTGCAAGGGATGCATG  120************************************************************ NC_004108TGTAGCCAAGGATATCGATGTTGACCTATTAATGGCCAGACATGATTATTTTGGTAGAGA  180EF485035 TGTAGCCAAGGATATCGATGTTGACCTATTAATGGCCAGACATGATTATTTTGGTAGAGA 180 ************************************************************NC_004108 GCTGTGCAAGTCCTTAAATATAGAATATAGGAATGATGTACCATTTGTAGATATAATTTT 240 EF485035GCTGTGCAAGTCCTTAAATATAGAATATAGGAATGATGTACCATTTGTAGATATAATTTT  240************************************************************ NC_004108GGATATAAGGCCCGAAGTAGACCCATTAACCATAGATGCACCACATATTACCCCAGACAA  300EF485035 GGATATAAGGCCCGAAGTAGACCCATTAACCATAGATGCACCACATATTACCCCAGACAA 300 ************************************************************NC_004108 TTATCTATATATAAATAATGTGTTATATATCATAGATTATAAGGTCTCTGTATCGAATGA 360 EF485035TTATCTATATATAAATAATGTGTTATATATCATAGATTATAAGGTCTCTGTATCGAATGA  360************************************************************ NC_004108AAGCAGTGTTATAACATATGACAAATATTATGAGTTAACTAGGGACATATCCGATAGATT  420EF485035 AAGCAGTGTTATAACATATGACAAATATTATGAGTTAACTAGGGACATATCCGATAGATT 420 ************************************************************NC_004108 AAGTATTCCAATAGAAATAGTTATCGTCCGTATAGACCCTGTAAGTAAGGATTTGCATAT 480 EF485035AAGTATTCCAATAGAAATAGTTATCGTCCGTATAGACCCTGTAAGTAAGGATTTGCATAT  480************************************************************ NC_004108TAACTCTGATAGATTTAAAGAACTTTACCCTACAATAGTGGTGGATATAAACTTCAATCA  540EF485035 TAACTCTGATAGATTTAAAGAACTTTACCCTACAATAGTGGTGGATATAAACTTCAATCA 540 ************************************************************NC_004108 ATTTTTCGACTTAAAACAATTACTCTATGAAAAATTCGGTGATGATGAAGAATTCCTATT 600 EF485035ATTTTTCGACTTAAAACAATTACTCTATGAAAAATTCGGTGATGATGAAGAATTCCTATT  600************************************************************ NC_004108GAAAGTTGCACATGGTGACTTCACTCTTACAGCACCCTGGTGCAAGACTGGGTGCCCTGA  660EF485035 GAAAGTTGCACATGGTGACTTCACTCTTACAGCACCCTGGTGCAAGACTGGGTGCCCTGA 660 ************************************************************NC_004108 ATTTTGGAAACACCCCATTTATAAAGAATTTAAAATGAGTATGCCAGTACCTGAGCGGAG 720 EF485035ATTTTGGAAACACCCCATTTATAAAGAATTTAAAATGAGTATGCCAGTACCTGAGCGGAG  720************************************************************ NC_004108GCTCTTTGAAGAATCTGTCAAGTTCAATGCTTATGAATCTGAGAGATGGAATACTAACTT  780EF485035 GCTCTTTGAAGAATCTGTCAAGTTCAATGCTTATGAATCTGAGAGATGGAATACTAACTT 780 ************************************************************NC_004108 GGTTAAAATCAGAGAATATACAAAGAAAGACTATTCAGAGCATATTTCAAAATCTGCAAA 840 EF485035GGTTAAAATCAGAGAATATACAAAGAAAGACTATTCAGAGCATATTTCAAAATCTGCAAA  840************************************************************ NC_004108AAATATTTTCCTGGCTAGTGGATTTTATAACCAGCCAAATAAGAATGAGATTAGTGAGGG  900EF485035 AAATATTTTCCTGGCTAGTGGATTTTATAACCAGCCAAATAAGAATGAGATTAGTGAGGG 900 ************************************************************NC_004108 GTGGACATTAATGGTTGAGAGGGTTCAAGATCAGAGAGAAATCTCAAAATCTCTCCATGA 960 EF485035GTGGACATTAATGGTTGAGAGGGTTCAAGATCAGAGAGAAATCTCAAAATCTCTCCATGA  960************************************************************ NC_004108CCAGAAACCTAGCATACATTTTATATGGGGAGCCCATAACCCAGGAAATAGTAATAATGC 1020EF485035 CCAGAAACCTAGCATACATTTTATATGGGGAGCCCATAACCCAGGAAATAGTAATAATGC1020 ************************************************************NC_004108 AACCTTCAAACTCATATTGCTTTCAAAGTCCTTACAAAGCATAAAAGGTATATCAACTTA1080 EF485035AACCTTCAAACTCATATTGCTTTCAAAGTCCTTACAAAGCATAAAAGGTATATCAACTTA 1080************************************************************ NC_004108CACAGAAGCGTTCAAATCTTTAGGAAAAATGATGGATATTGGAGATAAGGCTATTGAGTA 1140EF485035 CACAGAAGCGTTCAAATCTTTAGGAAAAATGATGGATATTGGAGATAAGGCTATTGAGTA1140 ************************************************************NC_004108 TGAAGAATTCTGCATGTCCCTAAAAAGCAAAGCAAGATCATCATGGAAGCAAATAATGAA1200 EF485035TGAAGAATTCTGCATGTCCCTAAAAAGCAAAGCAAGATCATCATGGAAGCAAATAATGAA 1200************************************************************ NC_004108CAAAAAATTAGAGCCTAAACAAATAAACAATGCCCTTGTTTTATGGGAACAGCAGTTTAT 1260EF485035 CAAAAAATTAGAGCCTAAACAAATAAACAATGCCCTTGTTTTATGGGAACAGCAGTTTAT1260 ************************************************************NC_004108 GGTAAATAATGACCTGATAGACAAAAGTGAGAAGTTGAAATTATTCAAAAATTTCTGCGG1320 EF485035GGTAAATAATGACCTGATAGACAAAAGTGAGAAGTTGAAATTATTCAAAAATTTCTGCGG 1320************************************************************ NC_004108TATAGGCAAACACAAGCAATTCAAGAATAAAATGCTAGAGGATCTAGAAGTGTCAAAGCC 1380EF485035 TATAGGCAAACACAAGCAATTCAAGAATAAAATGCTAGAGGATCTAGAAGTGTCAAAGCC1380 ************************************************************NC_004108 CAAAATATTAGACTTTGATGACGCAAATATGTATCTAGCTAGCCTAACCATGATGGAACA1440 EF485035CAAAATATTAGACTTTGATGACGCAAATATGTATCTAGCTAGCCTAACCATGATGGAACA 1440************************************************************ NC_004108GAGTAAGAAGATATTGTCCAAAAGCAATGGGTTGAAGCCAGATAATTTTATACTGAATGA 1500EF485035 GAGTAAGAAGATATTGTCCAAAAGCAATGGGTTGAAGCCAGATAATTTTATACTGAATGA1500 ************************************************************NC_004108 ATTTGGATCCAAAATCAAAGATGCTAATAAAGAAACATATGACAATATGCACAAAATATT1560 EF485035ATTTGGATCCAAAATCAAAGATGCTAATAAAGAAACATATGACAATATGCACAAAATATT 1560************************************************************ NC_004108TGAGACAAGATATTGGCAATGTATATCCGACTTCTCTACTCTGATGAAAAATATCTTATC 1620EF485035 TGAGACAAGATATTGGCAATGTATATCCGACTTCTCTACTCTGATGAAAAATATCTTATC1620 ************************************************************NC_004108 TGTGTCCCAATATAACAGGCACAACACATTTAGGATAGCTATGTGTGCTAATAACAATGT1680 EF485035TGTGTCCCAATATAACAGGCACAACACATTTAGGATAGCTATGTGTGCTAATAACAATGT 1680************************************************************ NC_004108CTTTGCTATAGTATTTCCTTCGGCTGACATAAAAACTAAGAAAGCAACTGTAGTTTATAG 1740EF485035 CTTTGCTATAGTATTTCCTTCGGCTGACATAAAAACTAAGAAAGCAACTGTAGTTTATAG1740 ************************************************************NC_004108 CATTATAGTGCTGCATAAAGAGGAAGAAAACATATTCAACCCAGGATGTTTGCACGGCAC1800 EF485035CATTATAGTGCTGCATAAAGAGGAAGAAAACATATTCAACCCAGGATGTTTGCACGGCAC 1800************************************************************ NC_004108ATTTAAGTGTATGAATGGGTATATTTCCATATCTAGAGCTATAAGGCTAGATAAAGAGAG 1860EF485035 ATTTAAGTGTATGAATGGGTATATTTCCATATCTAGAGCTATAAGGCTAGATAAAGAGAG1860 ************************************************************NC_004108 GTGCCAGAGAATTGTTTCCTCACCTGGACTGTTTTTAACAACTTGCCTACTATTCAAACA1920 EF485035GTGCCAGAGAATTGTTTCCTCACCTGGACTGTTTTTAACAACTTGCCTACTATTCAAACA 1920************************************************************ NC_004108TGATAATCCAACTCTAGTGATGAGCGATATTATGAATTTTTCTATATACACTAGCCTGTC 1980EF485035 TGATAATCCAACTCTAGTGATGAGCGATATTATGAATTTTTCTATATACACTAGCCTGTC1980 ************************************************************NC_004108 TATCACAAAGAGTGTTCTATCTTTAACAGAGCCAGCACGCTACATGATTATGAACTCATT2040 EF485035TATCACAAAGAGTGTTCTATCTTTAACAGAGCCAGCACGCTACATGATTATGAACTCATT 2040************************************************************ NC_004108AGCTATCTCCAGCAATGTTAAGGACTATATAGCAGAGAAATTTTCCCCTTACACAAAGAC 2100EF485035 AGCTATCTCCAGCAATGTTAAGGACTATATAGCAGAGAAATTTTCCCCTTACACAAAGAC2100 ************************************************************NC_004108 ACTGTTCAGTGTCTATATGACTAGACTAATTAAAAATGCTTGCTTTGATGCTTATGACCA2160 EF485035ACTGTTCAGTGTCTATATGACTAGACTAATTAAAAATGCTTGCTTTGATGCTTATGACCA 2160************************************************************ NC_004108GAGACAGCGTGTCCAACTTAGAGATATATATTTATCTGATTATGACATAACCCAAAAAGG 2220EF485035 GAGACAGCGTGTCCAACTTAGAGATATATATTTATCTGATTATGACATAACCCAAAAAGG2220 ************************************************************NC_004108 TATTAAAGACAATAGAGAGCTAACAAGTATATGGTTCCCTGGTAGTGTAACATTAAAGGA2280 EF485035TATTAAAGACAATAGAGAGCTAACAAGTATATGGTTCCCTGGTAGTGTAACATTAAAGGA 2280************************************************************ NC_004108GTATTTAACACAAATATACTTACCATTTTATTTTAATGCTAAAGGACTACATGAGAAGCA 2340EF485035 GTATTTAACACAAATATACTTACCATTTTATTTTAATGCTAAAGGACTACATGAGAAGCA2340 ************************************************************NC_004108 CCATGTCATGGTGGATCTAGCAAAGACTATATTAGAAATAGAGTGCGAACAGAGGGAAAA2400 EF485035CCATGTCATGGTGGATCTAGCAAAGACTATATTAGAAATAGAGTGCGAACAGAGGGAAAA 2400************************************************************ NC_004108CATAAAGGAGATATGGTCTACAAATTGTACCAAACAGACAGTGAACCTTAAAATTTTGAT 2460EF485035 CATAAAGGAGATATGGTCTACAAATTGTACCAAACAGACAGTGAACCTTAAAATTTTGAT2460 ************************************************************NC_004108 CCATTCCTTGTGCAAGAATTTACTAGCAGACACTTCAAGACACAACCACTTGCGGAACAG2520 EF485035CCATTCCTTGTGCAAGAATTTACTAGCAGACACTTCAAGACACAACCACTTGCGGAACAG 2520************************************************************ NC_004108AATAGAAAATAGGAACAATTTTAGAAGGTCTATAACAACTATTTCAACATTTACAAGTTC 2580EF485035 AATAGAAAATAGGAACAATTTTAGAAGGTCTATAACAACTATTTCAACATTTACAAGTTC2580 ************************************************************NC_004108 AAAGTCTTGCCTCAAAATAGGGGACTTTAGAAAAGAGAAAGAGCTGCAGTCAGTTAAACA2640 EF485035GAAGTCTTGCCTCAAAATAGGGGACTTTAGAAAAGAGAAAGAGCTGCAGTCAGTTAAACA 2640 *********************************************************** NC_004108GAAGAAAATCTTAGAGGTGCAGAGTCGCAAAATGAGATTAGCAAACCCAATGTTCGTGAC 2700EF485035 GAAGAAAATCTTAGAGGTGCAGAGTCGCAAAATGAGATTAGCAAACCCAATGTTCGTGAC2700 ************************************************************NC_004108 AGATGAACAAGTATGCCTTGAAGTTGGGCACTGCAATTATGAGATGCTGAGGAATGCTAT2760 EF485035AGATGAACAAGTATGCCTTGAAGTTGGGCACTGCAATTATGAGATGCTGAGGAATGCTAT 2760************************************************************ NC_004108GCCGAATTATACAGATTATATATCAACTAAAGTATTTGATAGGTTATATGAGTTATTAGA 2820EF485035 GCCGAATTATACAGATTATATATCAACTAAAGTATTTGATAGGTTATATGAGTTATTAGA2820 ************************************************************NC_004108 TAAAGGAGTTTTGACAGACAAGCCTGTTATAGAGCAAATAATGGATATGATGGTCGACCA2880 EF485035TAAAGGAGTTTTGACAGACAAGCCTGTTATAGAGCAAATAATGGATATGATGGTCGACCA 2880************************************************************ NC_004108CAAAAAGTTCTATTTCACATTTTTCAATAAAGGCCAGAAAACGTCAAAGGATAGAGAGAT 2940EF485035 CAAAAAGTTCTATTTCACATTTTTCAATAAAGGCCAGAAAACGTCAAAGGATAGAGAGAT2940 ************************************************************NC_004108 ATTCGTTGGAGAATATGAAGCTAAAATGTGTATGTACGCAGTTGAGAGAATAGCAAAAGA3000 EF485035ATTCGTTGGAGAATATGAAGCTAAAATGTGTATGTACGCAGTTGAGAGAATAGCAAAAGA 3000************************************************************ NC_004108AAGATGTAAATTAAATCCTGATGAAATGATATCTGAGCCGGGTGATGGCAAGTTGAAGGT 3060EF485035 AAGATGTAAATTAAATCCTGATGAAATGATATCTGAGCCGGGTGATGGCAAGTTGAAGGT3060 ************************************************************NC_004108 GTTGGAGCAAAAATCAGAACAAGAAATTCGATTCTTGGTCGAGACTACAAGGCAAAAGAA3120 EF485035GTTGGAGCAAAAATCAGAACAAGAAATTCGATTCTTGGTCGAGACTACAAGGCAAAAGAA 3120************************************************************ NC_004108TCGTGAAATCGATGAGGCAATTGAAGCATTAGCTGCAGAAGGATATGAGAGTAATCTAGA 3180EF485035 TCGTGAAATCGATGAGGCAATTGAAGCATTAGCTGCAGAAGGATATGAGAGTAATCTAGA3180 ************************************************************NC_004108 AAAAATTGAAAAGCTTTCACTTGGCAAAGCAAAGGGCCTAAAGATGGAAATAAATGCAGA3240 EF485035AAAAATTGAAAAGCTTTCACTTGGCAAAGCAAAGGGCCTAAAGATGGAAATAAATGCAGA 3240************************************************************ NC_004108TATGTCTAAATGGAGTGCTCAGGATGTTTTTTATAAATATTTCTGGCTCATAGCCTTAGA 3300EF485035 TATGTCTAAATGGAGTGCTCAGGATGTTTTTTATAAATATTTCTGGCTCATAGCCTTAGA3300 ************************************************************NC_004108 CCCTATCCTCTACCCACAGGAAAAAGAGAGAATATTATACTTTATGTGCAACTACATGGA3360 EF485035CCCTATCCTCTACCCACAGGAAAAAGAGAGAATATTATACTTTATGTGCAACTACATGGA 3360************************************************************ NC_004108TAAAGAATTGATACTGCCAGATGAATTATTATTCAATTTGCTGGACCAAAAAGTTGCATA 3420EF485035 TAAAGAATTGATACTGCCAGATGAATTATTATTCAATTTGCTGGACCAAAAAGTTGCATA3420 ************************************************************NC_004108 CCAGAATGATATAATAGCTACTATGACTAATCAATTAAATTCAAATACAGTTCTGATAAA3480 EF485035CCAGAATGATATAATAGCTACTATGACTAATCAATTAAATTCAAATACAGTTCTGATAAA 3480************************************************************ NC_004108GAGAAATTGGCTCCAAGGGAATTTCAACTACACCTCAAGTTACGTCCATAGCTGCGCAAT 3540EF485035 GAGAAATTGGCTCCAAGGGAATTTCAACTACACCTCAAGTTACGTCCATAGCTGCGCAAT3540 ************************************************************NC_004108 GTCTGTGTATAAAGAAATATTAAAAGAGGCCATAACATTACTAGACGGGTCTATATTAGT3600 EF485035GTCTGTGTATAAAGAAATATTAAAAGAGGCCATAACATTACTAGACGGGTCTATATTAGT 3600************************************************************ NC_004108CAACTCATTAGTCCATTCGGATGATAACCAAACATCGATAACAATAGTTCAGGATAAGAT 3660EF485035 CAACTCATTAGTCCATTCGGATGATAACCAAACATCGATAACAATAGTTCAGGATAAGAT3660 ************************************************************NC_004108 GGAAAATGATAAAATTATAGATTTTGCAATGAAAGAATTTGAGAGAGCCTGTTTGACATT3720 EF485035GGAAAATGATAAAATTATAGATTTTGCAATGAAAGAATTTGAGAGAGCCTGTTTGACATT 3720************************************************************ NC_004108TGGATGCCAAGCAAATATGAAAAAGACATATGTAACAAATTGCATAAAAGAGTTTGTTTC 3780EF485035 TGGATGCCAAGCAAATATGAAAAAGACATATGTAACAAATTGCATAAAAGAGTTTGTTTC3780 ************************************************************NC_004108 ATTATTTAACTTGTACGGCGAACCCTTTTCAATATATGGCAGATTCCTATTAACATCTGT3840 EF485035ATTATTTAACTTGTACGGCGAACCCTTTTCAATATATGGCAGATTCCTATTAACATCTGT 3840************************************************************ NC_004108GGGTGATTGTGCCTATATAGGGCCTTATGAAGATTTAGCTAGTCGAATATCATCAGCCCA 3900EF485035 GGGTGATTGTGCCTATATAGGGCCTTATGAAGATTTAGCTAGTCGAATATCATCAGCCCA3900 ************************************************************NC_004108 GACAGCCATAAAGCATGGTTGTCCACCCAGTCTAGCATGGGTGTCCATAGCAATAAGTCA3960 EF485035GACAGCCATAAAGCATGGTTGTCCACCCAGTCTAGCATGGGTGTCCATAGCAATAAGTCA 3960************************************************************ NC_004108TTGGATGACCTCTCTGACATACAACATGCTACCAGGGCAGTCAAATGACCCAATTGATTA 4020EF485035 TTGGATGACCTCTCTGACATACAACATGCTACCAGGGCAGTCAAATGACCCAATTGATTA4020 ************************************************************NC_004108 TTTCCCTGCAGAAAATAGGAAGGATATCCCTATAGAATTGAATGGTGTATTAGATGCTCC4080 EF485035TTTCCCTGCAGAAAATAGGAAGGATATCCCTATAGAATTGAATGGTGTATTAGATGCTCC 4080************************************************************ NC_004108ATTGTCAATGATTAGTACAGTTGGATTGGAATCTGGGAATTTATACTTCTTGATAAAGTT 4140EF485035 ATTGTCAATGATTAGTACAGTTGGATTGGAATCTGGGAATTTATACTTCTTGATAAAGTT4140 ************************************************************NC_004108 GTTGAGCAAATATACCCCGGTCATGCAGAAAAGAGAGTCAGTAGTCAACCAAATAGCTGA4200 EF485035GTTGAGCAAATATACCCCGGTCATGCAGAAAAGAGAGTCAGTAGTCAACCAAATAGCTGA 4200************************************************************ NC_004108AGTTAAGAACTGGAAGGTCGAGGATCTAACAGACAATGAAATATTTAGACTTAAAATACT 4260EF485035 AGTTAAGAACTGGAAGGTCGAGGATCTAACAGACAATGAAATATTTAGACTTAAAATACT4260 ************************************************************NC_004108 CAGATATTTAGTTCTAGATGCAGAGATGGACCCTAGTGATATTATGGGTGAGACAAGCGA4320 EF485035CAGATATTTAGTTCTAGATGCAGAGATGGACCCTAGTGATATTATGGGTGAGACAAGCGA 4320************************************************************ NC_004108CATGAGAGGGAGGTCTATTTTGACACCTAGAAAATTCACAACAGCAGGCAGTTTAAGGAA 4380EF485035 CATGAGAGGGAGGTCTATTTTGACACCTAGAAAATTCACAACAGCAGGCAGTTTAAGGAA4380 ************************************************************NC_004108 ATTATATTCTTTCAGTAAGTACCAGGATAGACTGTCTTCCCCTGGAGGCATGGTTGAATT4440 EF485035ATTATATTCTTTCAGTAAGTACCAGGATAGACTGTCTTCCCCTGGAGGCATGGTTGAATT 4440************************************************************ NC_004108GTTCACTTATTTGCTTGAGAAACCTGAGTTGTTAGTGACTAAAGGGGAAGATATGAAAGA 4500EF485035 GTTCACTTATTTGCTTGAGAAACCTGAGTTGTTAGTGACTAAAGGGGAAGATATGAAAGA4500 ************************************************************NC_004108 TTATATGGAATCTGTGATATTCCGATATAATTCCAAAAGGTTCAAAGAAAGTTTGTCAAT4560 EF485035TTATATGGAATCTGTGATATTCCGATATAATTCCAAAAGGTTCAAAGAAAGTTTGTCAAT 4560************************************************************ NC_004108ACAGAACCCAGCACAATTATTTATAGAACAGATATTGTTCTCACATAAGCCCATAATAGA 4620EF485035 ACAGAACCCAGCACAATTATTTATAGAACAGATATTGTTCTCACATAAGCCCATAATAGA4620 ************************************************************NC_004108 CTTTTCTGGTATCAGGGACAAATATATAAACCTACATGATAGTAGAGCTCTAGAGAAGGA4680 EF485035CTTTTCTGGTATCAGGGACAAATATATAAACCTACATGATAGTAGAGCTCTAGAGAAGGA 4680************************************************************ NC_004108ACCTGACATATTAGGAAAAGTAACATTTACAGAGGCTTATAGATTATTAATGAGGGACCT 4740EF485035 ACCTGACATATTAGGAAAAGTAACATTTACAGAGGCTTATAGATTATTAATGAGGGACCT4740 ************************************************************NC_004108 GTCTAGCCTAGAACTAACCAATGATGACATTCAAGTAATTTATTCTTACATAATACTTAA4800 EF485035GTCTAGCCTAGAACTAACCAATGATGACATTCAAGTAATTTATTCTTACATAATACTTAA 4800************************************************************ NC_004108TGACCCTATGATGATAACTATTGCAAACACACATATATTGTCAATATACGGGAGTCCTCA 4860EF485035 TGACCCTATGATGATAACTATTGCAAACACACATATATTGTCAATATACGGGAGTCCTCA4860 ************************************************************NC_004108 ACGGAGGATGGGCATGTCCTGTTCAACGATGCCAGAATTTAGAAATTTAAAATTAATACA4920 EF485035ACGGAGGATGGGCATGTCCTGTTCAACGATGCCAGAATTTAGAAATTTAAAATTAATACA 4920************************************************************ NC_004108TCATTCCCCAGCCTTAGTTTTGAGAGCATATAGTAAAAATAATCCTGACATCCAGGGTGC 4980EF485035 TCATTCCCCAGCCTTAGTTTTGAGAGCATATAGTAAAAATAATCCTGACATCCAGGGTGC4980 ************************************************************NC_004108 TGATCCCACGGAAATGGCTAGAGATTTAGTTCATCTGAAAGAGTTTGTTGAGAACACAAA5040 EF485035TGATCCCACGGAAATGGCTAGAGATTTAGTTCATCTGAAAGAGTTTGTTGAGAACACAAA 5040************************************************************ NC_004108TTTAGAAGAAAAAATGAAAGTTAGGATTGCTATAAATGAAGCAGAGAAAGGACAACGGGA 5100EF485035 TTTAGAAGAAAAAATGAAAGTTAGGATTGCTATAAATGAAGCAGAGAAAGGACAACGGGA5100 ************************************************************NC_004108 TATAGTCTTTGAACTAAAAGAGATGACTAGATTTTATCAGGTTTGCTATGAGTATGTCAA5160 EF485035TATAGTCTTTGAACTAAAAGAGATGACTAGATTTTATCAGGTTTGCTATGAGTATGTCAA 5160************************************************************ NC_004108ATCTACAGAACACAAGATAAAAGTCTTCATTCTCCCGACAAAATCATACACAACAACAGA 5220EF485035 ATCTACAGAACACAAGATAAAAGTCTTCATTCTCCCGACAAAATCATACACAACAACAGA5220 ************************************************************NC_004108 TTTCTGTTCACTCATGCAGGGGAATTTAATAAAAGATAAAGAGTGGTACACAGTTCACTA5280 EF485035TTTCTGTTCACTCATGCAGGGGAATTTAATAAAAGATAAAGAGTGGTACACAGTTCACTA 5280************************************************************ NC_004108CCTAAAACAGATATTGTCTGGTGGCCATAAAGCCATAATGCAGCATAATGCCACTAGTGA 5340EF485035 CCTAAAACAGATATTGTCTGGTGGCCATAAAGCCATAATGCAGCATAATGCCACTAGTGA5340 ************************************************************NC_004108 GCAAAATATTGCTTTTGAGTGTTTCAAATTAATTACCCATTTTGCAGACTCATTCATAGA5400 EF485035GCAAAATATTGCTTTTGAGTGTTTCAAATTAATTACCCATTTTGCAGACTCATTCATAGA 5400************************************************************ NC_004108TTCATTATCTAGGTCAGCTTTTTTGCAGTTGATAATAGATGAATTCAGTTATAAAGATGT 5460EF485035 TTCATTATCTAGGTCAGCTTTTTTGCAGTTGATAATAGATGAATTCAGTTATAAAGATGT5460 ************************************************************NC_004108 GAAGGTTAGCAAACTTTATGACATAATAAAGAATGGGTATAATCGAACTGACTTCATACC5520 EF485035GAAGGTTAGCAAACTTTATGACATAATAAAGAATGGGTATAATCGAACTGACTTCATACC 5520************************************************************ NC_004108ATTGCTTTTTAGAACTGGCGATTTAAGACAAGCTGACTTAGACAAGTATGATGCTATGAA 5580EF485035 ATTGCTTTTTAGAACTGGCGATTTAAGACAAGCTGACTTAGACAAGTATGATGCTATGAA5580 ************************************************************NC_004108 AAGTCATGAGAGGGTTACATGGAATGATTGGCAAACATCTCGTCACTTGGACATGGGCTC5640 EF485035AAGTCATGAGAGGGTTACATGGAATGATTGGCAAACATCTCGTCACTTGGACATGGGCTC 5640************************************************************ NC_004108AATTAATCTAACAATAACCGGTTACAATAGATCAATAACAATAATCGGAGAAGATAACAA 5700EF485035 AATTAATCTAACAATAACCGGTTACAATAGATCAATAACAATAATCGGAGAAGATAACAA5700 ************************************************************NC_004108 ATTGACATATGCAGAATTATGTCTGACTAGGAAAACTCCTGAGAATATAACTATAAGTGG5760 EF485035ATTGACATATGCAGAATTATGTCTGACTAGGAAAACTCCTGAGAATATAACTATAAGTGG 5760************************************************************ NC_004108CAGAAAATTGCTAGGTGCAAGGCATGGACTTAAATTTGAAAATATGTCCAAAATCCAAAC 5820EF485035 CAGAAAATTGCTAGGTGCAAGGCATGGACTTAAATTTGAAAATATGTCCAAAATCCAAAC5820 ************************************************************NC_004108 ATACCCAGGCAATTATTATATAACATATAGAAAGAAAGATCGCCACCAGTTTGTATACCA5880 EF485035ATACCCAGGCAATTATTATATAACATATAGAAAGAAAGATCGCCACCAGTTTGTATACCA 5880************************************************************ NC_004108GATACATTCTCATGAATCAATAACAAGGAGGAATGAAGAGCATATGGCTATCAGGACCAG 5940EF485035 GATACATTCTCATGAATCAATAACAAGGAGGAATGAAGAGCATATGGCTATCAGGACCAG5940 ************************************************************NC_004108 AATATACAATGAAATAACTCCAGTATGTGTAGTTAACGTTGCAGAGGTGGATGGGGACCA6000 EF485035AATATACAATGAAATAACTCCAGTATGTGTAGTTAACGTTGCAGAGGTGGATGGGGACCA 6000************************************************************ NC_004108ACGTATATTGATAAGATCTTTAGACTATCTAAATAATGATATATTTTCTCTTTCAAGGAT 6060EF485035 ACGTATATTGATAAGATCTTTAGACTATCTAAATAATGATATATTTTCTCTTTCAAGGAT6060 ************************************************************NC_004108 TAAAGTCGGGCTTGACGAATTTGCAACAATAAAAAAAGCACACTTTAGTAAAATGGTCTC6120 EF485035TAAAGTCGGGCTTGACGAATTTGCAACAATAAAAAAAGCACACTTTAGTAAAATGGTCTC 6120************************************************************ NC_004108ATTTGAAGGACCCCCAATTAAGACAGGGCTCCTCGACCTTACTGAATTGATGAAATCTCA 6180EF485035 ATTTGAAGGACCCCCAATTAAGACAGGGCTCCTCGACCTTACTGAATTGATGAAATCTCA6180 ************************************************************NC_004108 AGATTTGCTTAACCTTAATTATGATAATATAAGGAATAGCAACTTGATATCTTTTTCAAA6240 EF485035AGATTTGCTTAACCTTAATTATGATAATATAAGGAATAGCAACTTGATATCTTTTTCAAA 6240************************************************************ NC_004108ATTGATTTGCTGTGAGGGGTCAGATAATATAAATGATGGGTTAGAGTTTCTGTCCGATGA 6300EF485035 ATTGATTTGCTGTGAGGGGTCAGATAATATAAATGATGGGTTAGAGTTTCTGTCCGATGA6300 ************************************************************NC_004108 CCCTATGAACTTTACAGAGGGTGAAGCAATACATTCAACACCGATCTTTAATATATATTA6360 EF485035CCCTATGAACTTTACAGAGGGTGAAGCAATACATTCAACACCGATCTTTAATATATATTA 6360************************************************************ NC_004108CTCAAAAAGAGGAGAAAGACATATGACATACAGGAATGCAATTAAATTACTGATAGAAAG 6420EF485035 CTCAAAAAGAGGAGAAAGACATATGACATACAGGAATGCAATTAAATTACTGATAGAAAG6420 ************************************************************NC_004108 AGAAACTAAGATTTTTGAAGAAGCTTTCACATTCAGTGAGAATGGCTTCATATCGCCAGA6480 EF485035AGAAACTAAGATTTTTGAAGAAGCTTTCACATTCAGTGAGAATGGCTTCATATCGCCAGA 6480************************************************************ NC_004108GAATCTTGGTTGCTTAGAAGCAGTAGTATCATTAATAAAATTGTTGAAAACTAATGAGTG 6540EF485035 GAATCTTGGTTGCTTAGAAGCAGTAGTATCATTAATAAAATTGTTGAAAACTAATGAGTG6540 ************************************************************NC_004108 GTCCACAGTTATAGATAAATGTATTCATATATGTTTAATAAAGAATGGTATGGATCACAT6600 EF485035GTCCACAGTTATAGATAAATGTATTCATATATGTTTAATAAAGAATGGTATGGATCACAT 6600************************************************************ NC_004108GTACCATTCATTTGATGTCCCTAAATGTTTTATGGGGAATCCTATCACTAGAGACATGAA 6660EF485035 GTACCATTCATTTGATGTCCCTAAATGTTTTATGGGGAATCCTATCACTAGAGACATGAA6660 ************************************************************NC_004108 TTGGATGATGTTTAGAGAATTCATCAATAGTTTACCAGGGACAGATATACCACCATGGAA6720 EF485035TTGGATGATGTTTAGAGAATTCATCAATAGTTTACCAGGGACAGATATACCACCATGGAA 6720************************************************************ NC_004108TGTCATGACAGAGAACTTCAAAAAGAAATGTATTGCTCTGATAAACTCTAAGTTAGAAAC 6780EF485035 TGTCATGACAGAGAACTTCAAAAAGAAATGTATTGCTCTGATAAACTCTAAGTTAGAAAC6780 ************************************************************NC_004108 ACAGAGAGATTTCTCAGAATTCACTAAACTGATGAAAAAGGAAGGTGGGAGGAGTAATAT6840 EF485035ACAGAGAGATTTCTCAGAATTCACTAAACTGATGAAAAAGGAAGGTGGGAGGAGTAATAT 6840************************************************************ NC_004108AGAATTTGATTAGTAGTTATGAGTTTACAGAGAACCTACAATTAGGCTATAAATTTGGGA 6900EF485035 AGAATTTGATTAGTAGTTATGAGTTTACAGAGAACCTACAATTAGGCTATAAATTTGGGA6900 ************************************************************NC_004108 GGGTTTTGGAAATTGGCTAAAATTCAAAAAGAGGGGGATTAACAGCAACTGTATAAATTT6960 EF485035GGGTTTTGGAAATTGGCTAAAATTCAAAAAGAGGGGGATTAACAGCAACTGTATAAATTT 6960************************************************************ NC_004108GTAGATAGGGGCACACTACT (SEQ. ID. No. 1) 6980 EF485035GTAGATAGGAGCACACTACT (SEQ. ID. NO. 2) 6980 ********* **********

Appendix 2 Medium genomic segment-nucleotide sequence alignment ofLACV/human/1978 (Hughes et al. 2002) and LACV/human/1978 (this study)NC_004109 AGTAGTGTACTACCAAGTATAGATAACGTTTGAATATTAAAGTTTTGAATCAAAGCCAAA  60 EF485034AGTAGTGTACTACCAAGTATAGATAACGTTTGAATATTAAAGTTTTGAATCAAAGCCAAA   60************************************************************ NC_004109GATGATTTGTATATTGGTGCTAATTACAGTTGCAGCTGCAAGCCCAGTGTATCAAAGGTG  120EF485034 GATGATTTGTATATTGGTGCTAATTACAGTTGCAGCTGCAAGCCCAGTGTATCAAAGGTG 120 ************************************************************NC_004109 TTTCCAAGATGGGGCTATAGTGAAGCAAAACCCATCCAAAGAAGCAGTTACAGAGGTGTG 180 EF485034TTTCCAAGATGGGGCTATAGTGAAGCAAAACCCATCCAAAGAAGCAGTTACAGAGGTGTG  180************************************************************ NC_004109CCTGAAAGATGATGTTAGCATGATCAAAACAGAGGCCAGGTATGTAAGAAATGCAACAGG  240EF485034 CCTGAAAGATGATGTTAGCATGATCAAAACAGAGGCCAGGTATGTAAGAAATGCAACAGG 240 ************************************************************NC_004109 AGTTTTTTCAAATAATGTCGCAATAAGGAAATGGCTAGTCTCTGATTGGCATGATTGCAG 300 EF485034AGTTTTTTCAAATAATGTCGCAATAAGGAAATGGCTAGTCTCTGATTGGCATGATTGCAG  300************************************************************ NC_004109GCCTAAGAAGATCGTTGGGGGACACATCAATGTAATAGAAGTTGGTGATGACCTGTCACT  360EF485034 GCCTAAGAAGATCGTTGGGGGACACATCAATGTAATAGAAGTTGGTGATGACCTGTCACT 360 ************************************************************NC_004109 CCATACTGAATCATATGTTTGCAGCGCAGATTGTACCATAGGTGTAGACAAAGAGACTGC 420 EF485034CCATACTGAATCATATGTTTGCAGCGCAGATTGTACCATAGGTGTAGACAAAGAGACTGC  420************************************************************ NC_004109ACAGGTCAGGCTTCAGACAGATACCACAAATCATTTTGAAATTGCAGGCACTACTGTGAA  480EF485034 ACAGGTCAGGCTTCAGACAGATACCACAAATCATTTTGAAATTGCAGGCACTACTGTGAA 480 ************************************************************NC_004109 GTCAGGATGGTTCAAGAGCACGACATATATAACTCTTGATCAAACTTGCGAACACCTTAA 540 EF485034GTCAGGATGGTTCAAGAGCACGACATATATAACTCTTGATCAAACTTGCGAACACCTTAA  540************************************************************ NC_004109AGTTTCCTGCGGCCCAAAATCTGTACAGTTCCATGCCTGCTTCAATCAGCATATGTCTTG  600EF485034 AGTTTCCTGCGGCCCAAAATCTGTACAGTTCCATGCCTGCTTCAATCAGCATATGTCTTG 600 ************************************************************NC_004109 CGTCAGATTTTTACACAGGACAATATTGCCTGGCTCTATAGCCAATTCCATATGTCAGAA 660 EF485034CGTCAGATTTTTACACAGGACAATATTGCCTGGCTCTATAGCCAATTCCATATGTCAGAA  660************************************************************ NC_004109TATCGAAATCATAATTTTAGTTACACTTACTCTATTAATCTTTATATTGTTAAGCATTTT  720EF485034 TATCGAAATCATAATTTTAGTTACACTTACTCTATTAATCTTTATATTGTTAAGCATTTT 720 ************************************************************NC_004109 AAGTAAGACTTATATATGTTATTTATTAATGCCTATATTCATCCCCATAGCATATATATA 780 EF485034AAGTAAGACTTATATATGTTATTTATTAATGCCTATATTCATCCCCATAGCATATATATA  780************************************************************ NC_004109CGGTATAATTTACAATAAGTCGTGCAAAAAATGCAAATTATGTGGCTTAGTGTATCATCC  840EF485034 CGGTATAATTTACAATAAGTCGTGCAAAAAATGCAAATTATGTGGCTTAGTGTATCATCC 840 ************************************************************NC_004109 ATTCACAGAGTGTGGCACACATTGTGTCTGTGGTGCCCGCTATGATACTTCAGATAGAAT 900 EF485034ATTCACAGAGTGTGGCACACATTGTGTCTGTGGTGCCCGCTATGATACTTCAGATAGAAT  900************************************************************ NC_004109GAAACTGCATAGAGCTTCTGGATTGTGCCCTGGTTATAAAAGCCTAAGAGCTGCCAGAGT  960EF485034 GAAACTGCATAGAGCTTCTGGATTGTGCCCTGGTTATAAAAGCCTAAGAGCTGCCAGAGT 960 ************************************************************NC_004109 CATGTGCAAGTCGAAAGGGCCTGCATCAATATTGTCTATAATTACTGCGGTACTGGTCTT1020 EF485034CATGTGCAAGTCGAAAGGGCCTGCATCAATATTGTCTATAATTACTGCGGTACTGGTCTT 1020************************************************************ NC_004109AACCTTTGTGACACCAATCAACTCCATGGTTTTAGGAGAGAGTAAAGAAACCTTTGAACT 1080EF485034 AACCTTTGTGACACCAATCAACTCCATGGTTTTAGGAGAGAGTAAAGAAACCTTTGAACT1080 ************************************************************NC_004109 TGAAGATCTTCCAGACGACATGTTGGAAATGGCATCGAGAATAAATTCTTATTATCTCAC1140 EF485034TGAAGATCTTCCAGACGACATGTTGGAAATGGCATCGAGAATAAATTCTTATTATCTCAC 1140************************************************************ NC_004109CTGTATCTTGAATTATGCTGTAAGCTGGGGTCTTGTTATCATTGGATTGTTGATCGGGCT 1200EF485034 CTGTATCTTGAATTATGCTGTAAGCTGGGGTCTTGTTATCATTGGATTGTTGATCGGGCT1200 ************************************************************NC_004109 GCTTTTTAAGAAATACCAGCACAGATTCTTAAATGTTTACGCAATGTACTGTGAAGAATG1260 EF485034GCTTTTTAAGAAATACCAGCACAGATTCTTAAATGTTTACGCAATGTACTGTGAAGAATG 1260************************************************************ NC_004109TGACATGTATCATGACAAGTCTGGGTTGAAAAGACATGGTGATTTCACCAACAAATGCAG 1320EF485034 TGACATGTATCATGACAAGTCTGGGTTGAAAAGACATGGTGATTTCACCAACAAATGCAG1320 ************************************************************NC_004109 ACAGTGCACATGTGGTCAATATGAAGATGCTGCAGGTTTGATGGCTCACAGGAAAACCTA1380 EF485034ACAGTGCACATGTGGTCAATATGAAGATGCTGCAGGTTTGATGGCTCACAGGAAAACCTA 1380************************************************************ NC_004109TAACTGCTTAGTGCAGTACAAAGCAAAGTGGATGATGAACTTCCTGATAATTTACATATT 1440EF485034 TAACTGCTTAGTGCAGTACAAAGCAAAGTGGATGATGAACTTCCTGATAATTTACATATT1440 ************************************************************NC_004109 CTTAATTTTGATCAAAGATTCTGCTATAGTTGTACAAGCTGCTGGAACTGACTTCACCAC1500 EF485034CTTAATTTTGATCAAAGATTCTGCTATAGTTGTACAAGCTGCTGGAACTGACTTCACCAC 1500************************************************************ NC_004109CTGCCTAGAGACTGAGAGTATAAATTGGAACTGCACTGGGCCATTTTTGAACCTCGGGAA 1560EF485034 CTGCCTAGAGACTGAGAGTATAAATTGGAACTGCACTGGGCCATTTTTGAACCTCGGGAA1560 ************************************************************NC_004109 TTGCCAAAAGCAACAAAAGAAAGAACCTTACACCAACATTGCAACTCAGTTAAAGGGACT1620 EF485034TTGCCAAAAGCAACAAAAGAAAGAACCTTACACCAACATTGCAACTCAGTTAAAGGGACT 1620************************************************************ NC_004109AAAGGCAATTTCCGTACTAGATGTCCCTATAATAACAGGGATACCAGATGATATTGCGGG 1680EF485034 AAAGGCAATTTCCGTACTAGATGTCCCTATAATAACAGGGATACCAGATGATATTGCGGG1680 ************************************************************NC_004109 TGCTTTAAGATATATAGAAGAGAAGGAAGATTTCCATGTCCAGCTAACTATAGAATATGC1740 EF485034TGCTTTAAGATATATAGAAGAGAAGGAAGATTTCCATGTCCAGCTAACTATAGAATATGC 1740************************************************************ NC_004109GATGTTAAGCAAATACTGTGACTATTATACCCAATTCTCAGATAACTCAGGATACAGTCA 1800EF485034 GATGTTAAGCAAATACTGTGACTATTATACCCAATTCTCAGATAACTCAGGATACAGTCA1800 ************************************************************NC_004109 GACAACATGGAGAGTGTACTTAAGGTCTCATGATTTTGAAGCCTGTATACTATATCCAAA1860 EF485034GACAACATGGAGAGTGTACTTAAGGTCTCATGATTTTGAAGCCTGTATACTATATCCAAA 1860************************************************************ NC_004109TCAGCACTTTTGCAGATGTGTAAAAAATGGTGAGAAGTGCAGCAGCTCCAATTGGGACTT 1920EF485034 TCAGCACTTTTGCAGATGTGTAAAAAATGGTGAGAAGTGCAGCAGCTCCAATTGGGACTT1920 ************************************************************NC_004109 TGCCAATGAAATGAAAGATTATTACTCTGGGAAACAAACAAAGTTTGACAAGGACTTAAA1980 EF485034TGCCAATGAAATGAAAGATTATTACTCTGGGAAACAAACAAAGTTTGACAAGGACTTAAA 1980************************************************************ NC_004109TCTAGCCCTAACAGCTTTGCATCATGCCTTCAGGGGGACCTCATCTGCATATATAGCAAC 2040EF485034 TCTAGCCCTAACAGCTTTGCATCATGCCTTCAGGGGGACCTCATCTGCATATATAGCAAC2040 ************************************************************NC_004109 AATGCTCTCAAAAAAGTCCAATGATGACTTGATTGCATACACAAATAAGATAAAAACAAA2100 EF485034AATGCTCTCAAAAAAGTCCAATGATGACTTGATTGCATACACAAATAAGATAAAAACAAA 2100************************************************************ NC_004109ATTCCCAGGTAATGCATTGTTGAAGGCTATAATAGATTATATAGCATATATGAAAAGTTT 2160EF485034 ATTCCCAGGTAATGCATTGTTGAAGGCTATAATAGATTATATAGCATATATGAAAAGTTT2160 ************************************************************NC_004109 GCCAGGTATGGCAAATTTCAAATATGATGAATTCTGGGATGAATTACTGTACAAACCCAA2220 EF485034GCCAGGTATGGCAAATTTCAAATATGATGAATTCTGGGATGAATTACTGTACAAACCCAA 2220************************************************************ NC_004109CCCAGCAAAGGCCTCAAACCTTGCTAGAGGAAAGGAGTCATCTTACAACTTCAAACTAGC 2280EF485034 CCCAGCAAAGGCCTCAAACCTTGCTAGAGGAAAGGAGTCATCTTACAACTTCAAACTAGC2280 ************************************************************NC_004109 AATTTCATCAAAGTCTATAAAAACCTGCAAGAATGTTAAGGATGTTGCCTGCTTATCGCC2340 EF485034AATTTCATCAAAGTCTATAAAAACCTGCAAGAATGTTAAGGATGTTGCCTGCTTATCGCC 2340************************************************************ NC_004109AAGGTCAGGTGCTATATATGCTTCAATAATTGCGTGTGGTGAACCCAATGGGCCAAGTGT 2400EF485034 AAGGTCAGGTGCTATATATGCTTCAATAATTGCGTGTGGTGAACCCAATGGGCCAAGTGT2400 ************************************************************NC_004109 GTATAGGAAACCATCAGGTGGTGTATTCCAATCTAGCACTGATCGGTCTATATACTGCTT2460 EF485034GTATAGGAAACCATCAGGTGGTGTATTCCAATCTAGCACTGATCGGTCTATATACTGCTT 2460************************************************************ NC_004109GCTGGATAGCCATTGTCTAGAAGAATTTGAGGCCATCGGCCAGGAGGAGCTGGATGCGGT 2520EF485034 GCTGGATAGCCATTGTCTAGAAGAATTTGAGGCCATCGGCCAGGAGGAGCTGGATGCGGT2520 ************************************************************NC_004109 AAAGAAATCCAAATGTTGGGAAATTGAATATCCTGACGTAAAGCTCATCCAAGAAGGCGA2580 EF485034AAAGAAATCCAAATGTTGGGAAATTGAATATCCTGACGTAAAGCTCATCCAAGAAGGCGA 2580************************************************************ NC_004109TGGGACTAAAAGCTGTAGAATGAAAGATTCTGGGAACTGCAATGTTGCAACTAACAGATG 2640EF485034 TGGGACTAAAAGCTGTAGAATGAAAGATTCTGGGAACTGCAATGTTGCAACTAACAGATG2640 ************************************************************NC_004109 GCCAGTGATACAATGTGAGAATGACAAATTTTACTACTCAGAGCTTCAAAAAGATTATGA2700 EF485034GCCAGTGATACAATGTGAGAATGACAAATTTTACTACTCAGAGCTTCAAAAAGATTATGA 2700************************************************************ NC_004109CAAAGCTCAAGATATTGGTCACTATTGCTTAAGCCCTGGATGTACTACTGTCCGGTACCC 2760EF485034 CAAAGCTCAAGATATTGGTCACTATTGCTTAAGCCCTGGATGTACTACTGTCCGGTACCC2760 ************************************************************NC_004109 TATTAATCCAAAGCACATCTCTAACTGTAATTGGCAAGTAAGCAGATCTAGCATAGCGAA2820 EF485034TATTAATCCAAAGCACATCTCTAACTGTAATTGGCAAGTAAGCAGATCTAGCATAGCGAA 2820************************************************************ NC_004109GATAGATGTGCACAATATTGAGGATATTGAGCAATATAAGAAAGCTATAACTCAGAAACT 2880EF485034 GATAGATGTGCACAATATTGAGGATATTGAGCAATATAAGAAAGCTATAACTCAGAAACT2880 ************************************************************NC_004109 TCAAACGAGCCTATCTCTATTCAAGTATGCAAAAACAAAAAACTTGCCGCACATCAAACC2940 EF485034TCAAACGAGCCTATCTCTATTCAAGTATGCAAAAACAAAAAACTTGCCGCACATCAAACC 2940************************************************************ NC_004109AATTTATAAATATATAACTATAGAAGGAACAGAAACTGCAGAAGGTATAGAGAGTGCATA 3000EF485034 AATTTATAAATATATAACTATAGAAGGAACAGAAACTGCAGAAGGTATAGAGAGTGCATA3000 ************************************************************NC_004109 CATTGAATCAGAAGTACCTGCATTGGCTGGGACATCTATCGGATTCAAAATCAATTCTAA3060 EF485034CATTGAATCAGAAGTACCTGCATTGGCTGGGACATCTATCGGATTCAAAATCAATTCTAA 3060************************************************************ NC_004109AGAGGGCAAGCACTTGCTAGATGTTATAGCATATGTAAAAAGTGCCTCATACTCTTCAGT 3120EF485034 AGAGGGCAAGCACTTGCTAGATGTTATAGCATATGTAAAAAGTGCCTCATACTCTTCAGT3120 ************************************************************NC_004109 GTATACAAAATTGTACTCAACTGGCCCAACATCAGGGATAAATACTAAACATGATGAATT3180 EF485034GTATACAAAATTGTACTCAACTGGCCCAACATCAGGGATAAATACTAAACATGATGAATT 3180************************************************************ NC_004109GTGTACTGGCCCATGCCCAGCAAATATCAATCATCAGGTTGGGTGGCTGACATTTGCAAG 3240EF485034 GTGTACTGGCCCATGCCCAGCAAATATCAATCATCAGGTTGGGTGGCTGACATTTGCAAG3240 ************************************************************NC_004109 AGAGAGGACAAGCTCATGGGGATGCGAAGAGTTTGGTTGCCTGGCTGTAAGTGATGGGTG3300 EF485034AGAGAGGACAAGCTCATGGGGATGCGAAGAGTTTGGTTGCCTGGCTGTAAGTGATGGGTG 3300************************************************************ NC_004109TGTATTTGGATCATGCCAAGATATAATAAAAGAAGAACTATCTGTCTATAGGAAGGAGAC 3360EF485034 TGTATTTGGATCATGCCAAGATATAATAAAAGAAGAACTATCTGTCTATAGGAAGGAGAC3360 ************************************************************NC_004109 CGAGGAAGTGACTGATGTAGAACTGTGTTTGACATTTTCAGACAAAACATACTGTACAAA3420 EF485034CGAGGAAGTGACTGATGTAGAACTGTGTTTGACATTTTCAGACAAAACATACTGTACAAA 3420************************************************************ NC_004109CTTAAACCCTGTTACCCCTATTATAACAGATCTATTTGAGGTACAGTTCAAAACTGTAGA 3480EF485034 CTTAAACCCTGTTACCCCTATTATAACAGATCTATTTGAGGTACAGTTCAAAACTGTAGA3480 ************************************************************NC_004109 GACCTACAGCTTGCCTAGAATTGTTGCTGTGCAAAACCATGAGATTAAAATTGGGCAAAT3540 EF485034GACCTACAGCTTGCCTAGAATTGTTGCTGTGCAAAACCATGAGATTAAAATTGGGCAAAT 3540************************************************************ NC_004109AAATGATTTAGGAGTTTACTCTAAGGGTTGTGGGAATGTTCAAAAGGTCAATGGAACTAT 3600EF485034 AAATGATTTAGGAGTTTACTCTAAGGGTTGTGGGAATGTTCAAAAGGTCAATGGAACTAT3600 ************************************************************NC_004109 TTATGGCAATGGAGTTCCCAGATTTGACTACTTATGCCATTTAGCTAGCAGGAAGGAAGT3660 EF485034TTATGGCAATGGAGTTCCCAGATTTGACTACTTATGCCATTTAGCTAGCAGGAAGGAAGT 3660************************************************************ NC_004109CATTGTTAGAAAATGCTTCGACAATGATTACCAAGCATGCAAATTTCTTCAAAGCCCTGC 3720EF485034 CATTGTTAGAAAATGCTTCGACAATGATTACCAAGCATGCAAATTTCTTCAAAGCCCTGC3720 ************************************************************NC_004109 TAGTTACAGACTTGAAGAAGACAGTGGCACTGTGACCATAATTGACTACAAAAAGATTTT3780 EF485034TAGTTACAGACTTGAAGAAGACAGTGGCACTGTGACCATAATTGACTACAAAAAGATTTT 3780************************************************************ NC_004109AGGAACAATCAAGATGAAGGCAATTTTAGGAGATGTCAAATATAAAACATTTGCTGATAG 3840EF485034 AGGAACAATCAAGATGAAGGCAATTTTAGGAGATGTCAAATATAAAACATTTGCTGATAG3840 ************************************************************NC_004109 TGTCGATATAACCGCAGAAGGGTCATGCACCGGCTGTATTAACTGCTTCGAAAATATCCA3900 EF485034TGTCGATATAACCGCAGAAGGGTCATGCACCGGCTGTATTAACTGCTTCGAAAATATCCA 3900************************************************************ NC_004109TTGCGAATTAACGTTGCACACCACAATTGAAGCCAGCTGCCCAATTAAAAGCTCGTGCAC 3960EF485034 TTGCGAATTAACGTTGCACACCACAATTGAAGCCAGCTGCCCAATTAAAAGCTCGTGCAC3960 ************************************************************NC_004109 AGTATTTCATGACAGGATTCTTGTGACTCCAAATGAACACAAATATGCATTGAAAATGGT4020 EF485034AGTATTTCATGACAGGATTCTTGTGACTCCAAATGAACACAAATATGCATTGAAAATGGT 4020************************************************************ NC_004109GTGCACAGAAAAGCCAGGGAACACACTCACAATTAAAGTCTGCAATACTAAAGTTGAAGC 4080EF485034 GTGCACAGAAAAGCCAGGGAACACACTCACAATTAAAGTCTGCAATACTAAAGTTGAAGC4080 ************************************************************NC_004109 ATCTATGGCCCTTGTAGACGCAAAGCCTATCATAGAACTAGCACCAGTTGATCAGACAGC4140 EF485034ATCTATGGCCCTTGTAGACGCAAAGCCTATCATAGAACTAGCACCAGTTGATCAGACAGC 4140************************************************************ NC_004109ATATATAAGAGAAAAAGATGAAAGGTGTAAAACTTGGATGTGTAGGGTAAGAGATGAAGG 4200EF485034 ATATATAAGAGAAAAAGATGAAAGGTGTAAAACTTGGATGTGTAGGGTAAGAGATGAAGG4200 ************************************************************NC_004109 ACTGCAGGTCATCTTGGAGCCATTTAAAAATTTATTTGGATCTTATATTGGGATATTTTA4260 EF485034ACTGCAGGTCATCTTGGAGCCATTTAAAAATTTATTTGGATCTTATATTGGGATATTTTA 4260************************************************************ NC_004109CACATTTATTATATCTATAGTAGTATTATTGGTTATTATCTATGTACTACTACCTATATG 4320EF485034 CACATTTATTATATCTATAGTAGTATTATTGGTTATTATCTATGTACTACTACCTATATG4320 ************************************************************NC_004109 CTTTAAGTTAAGGGATACCCTTAGAAAGCATGAAGATGCATATAAGAGAGAGATGAAAAT4380 EF485034CTTTAAGTTAAGGGATACCCTTAGAAAGCATGAAGATGCATATAAGAGAGAGATGAAAAT 4380************************************************************ NC_004109TAGATAGGGGATCTATGCAGAACAAAATTGAGTCCTGTATTATATACTTCTATTTGTAGT 4440EF485034 TAGATAGGGGATCTATGCAGAACAAAATTGAGTCCTGTATTATATA-TTCTATTTGTAGT4439 ********************************************** *************NC_004109 ATAGCTGTTGTTAAGTGGGGGGTGGGGAACTAACAACAGCGTAAATTTATTTTGCAAACA4500 EF485034ATAGCTGTTGTTAAGTGGGGGGTGGGGAACTAACAACAGCGTAAATTTATTTTGCAAACA 4499************************************************************ NC_004109TTATTTTATACTTGGTAGCACACTACT (SEQ. ID. NO. 3) 4527 EF485034TTATTTTATACTTGGTAGCACACTACT (SEQ. ID. NO. 4) 4526***************************

APPENDIX 3Small genomic segment-nucleotide sequence alignment of LACV/human/1978(Hughes et al. 2002) and LACV/human/1978 (this study) NC_004110AGTAGTGTACCCCACTTGAATACTTTGAAAATAAATTGTTGTTGACTCTTTTTTACCTAA   60EF485033 AGTAGTGTACTCCACTTGAATACTTTGAAAATAAATTGTTGTTGACTCTTTTTTACCTAA  60 ************************************************************NC_004110 GGGGAAATTATCAAGAGTGTGATGTCGGATTTGGTGTTTTATGATGTCGCATCAACAGGT 120 EF485033GGGGAAATTATCAAGAGTGTGATGTCGGATTTGGTGTTTTATGATGTCGCATCAACAGGT  120************************************************************ NC_004110GCAAATGGATTTGATCCTGATGCAGGGTATATGGACTTCTGTGTTAAAAATGCAGAATTA  180EF485033 GCAAATGGATTTGATCCTGATGCAGGGTATATGGACTTCTGTGTTAAAAATGCAGAATTA 180 ************************************************************NC_004110 CTCAACCTTGCTGCAGTTAGGATCTTCTTCCTCAATGCCGCAAAGGCCAAGGCTGCTCTC 240 EF485033CTCAACCTTGCTGCAGTTAGGATCTTCTTCCTCAATGCCGCAAAGGCCAAGGCTGCTCTC  240************************************************************ NC_004110TCGCGTAAGCCAGAGAGGAAGGCTAACCCTAAATTTGGAGAGTGGCAGGTGGAGGTTATC  300EF485033 TCGCGTAAGCCAGAGAGGAAGGCTAACCCTAAATTTGGAGAGTGGCAGGTGGAGGTTATC 300 ************************************************************NC_004110 AATAATCATTTTCCTGGAAACAGGAACAACCCAATTGGTAACAACGATCTTACCATCCAC 360 EF485033AATAATCATTTTCCTGGAAACAGGAACAACCCAATTGGTAACAACGATCTTACCATCCAC  360************************************************************ NC_004110AGATTATCTGGGTATTTAGCCAGATGGGTCCTTGATCAGTATAACGAGAATGATGATGAG  420EF485033 AGATTATCTGGGTATTTAGCCAGATGGGTCCTTGATCAGTATAACGAGAATGATGATGAG 420 ************************************************************NC_004110 TCTCAGCACGAGTTGATCAGAACAACTATTATCAACCCAATTGCTGAGTCTAATGGTGTA 480 EF485033TCTCAGCACGAGTTGATCAGAACAACTATTATCAACCCAATTGCTGAGTCTAATGGTGTA  480************************************************************ NC_004110GGATGGGACAGTGGGCCAGAGATCTATCTATCATTCTTTCCAGGAACAGAAATGTTTTTG  540EF485033 GGATGGGACAGTGGGCCAGAGATCTATCTATCATTCTTTCCAGGAACAGAAATGTTTTTG 540 ************************************************************NC_004110 GAAACTTTCAAATTCTACCCGCTGACCATTGGAATTCACAGAGTCAAGCAAGGCATGATG 600 EF485033GAAACTTTCAAATTCTACCCGCTGACCATTGGAATTCACAGAGTCAAGCAAGGCATGATG  600************************************************************ NC_004110GACCCTCAATACCTGAAGAAGGCCTTAAGGCAACGCTATGGCACTCTCACAGCAGATAAG  660EF485033 GACCCTCAATACCTGAAGAAGGCCTTAAGGCAACGCTATGGCACTCTCACAGCAGATAAG 660 ************************************************************NC_004110 TGGATGTCACAGAAGGTTGCAGCAATTGCTAAGAGCCTGAAGGATGTAGAGCAGCTTAAA 720 EF485033TGGATGTCACAGAAGGTTGCAGCAATTGCTAAGAGCCTGAAGGATGTAGAGCAGCTTAAA  720************************************************************ NC_004110TGGGGAAAAGGAGGCCTGAGCGATACTGCTAAAACATTCCTGCAGAAATTTGGCATCAGG  780EF485033 TGGGGAAAAGGAGGCCTGAGCGATACTGCTAAAACATTCCTGCAGAAATTTGGCATCAGG 780 ************************************************************NC_004110 CTTCCATAAATATGGCATGAGGCATTCAAATTAGGTTCTAAATTCTAAATTTATATATGT 840 EF485033CTTCCATAAATATGGCATGAGGCATTCAAATTAGGTTCTAAATTCTAAATTTATATATGT  840************************************************************ NC_004110CAATTTGATTAATTGGTTATCCAAAAGGGTTTTCTTAAGGGAACCCACAAAAATAGCAGC  900EF485033 CAATTTGATTAATTGGTTATCCAAAAGGGTTTTCTTAAGGGAACCCACAAAAATAGCAGC 900 ************************************************************NC_004110 TAAATGGGTGGGTGGTAGGGGACAGCAAAAAACTATAAATCAGGTCATAAATAAAATAAA 960 EF485033TAAATGGGTGGGTGGTAGGGGACAGCAAAAAACTATAAATCAGGTCATAAATAAAATAAA  960************************************************************ NC_004110ATGTATTCAGTGGGGCACACTACT (SEQ. ID. No. 5)  984 EF485033ATGTATTCAGTGGAGCACACTACT (SEQ. ID. No. 6)  984 ************* **********

APPENDIX 4Large genomic segment-amino acid sequence alignment of LACV/human/1978(Hughes et al. 2002) and LACV/human/1978 (this study) NC_004108MDYQEYQQFLARINTARDACVAKDIDVDLLMARHDYFGRELCKSLNIEYRNDVPFVDIIL   60EF485035 MDYQEYQQFLARINTARDACVAKDIDVDLLMARHDYFGRELCKSLNIEYRNDVPFVDIIL  60 ************************************************************NC_004108 DIRPEVDPLTIDAPHITPDNYLYINNVLYIIDYKVSVSNESSVITYDKYYELTRDISDRL 120 EF485035DIRPEVDPLTIDAPHITPDNYLYINNVLYIIDYKVSVSNESSVITYDKYYELTRDISDRL  120************************************************************ NC_004108SIPIEIVIVRIDPVSKDLHINSDRFKELYPTIVVDINFNQFFDLKQLLYEKFGDDEEFLL  180EF485035 SIPIEIVIVRIDPVSKDLHINSDRFKELYPTIVVDINFNQFFDLKQLLYEKFGDDEEFLL 180 ************************************************************NC_004108 KVAHGDFTLTAPWCKTGCPEFWKHPIYKEFKMSMPVPERRLFEESVKFNAYESERWNTNL 240 EF485035KVAHGDFTLTAPWCKTGCPEFWKHPIYKEFKMSMPVPERRLFEESVKFNAYESERWNTNL  240************************************************************ NC_004108VKIREYTKKDYSEHISKSAKNIFLASGFYKQPNKNEISEGWTLMVERVQDQREISKSLHD  300EF485035 VKIREYTKKDYSEHISKSAKNIFLASGFYKQPNKNEISEGWTLMVERVQDQREISKSLHD 300 ************************************************************NC_004108 QKPSIHFIWGAHNPGNSNNATFKLILLSKSLQSIKGISTYTEAFKSLGKMMDIGDKAIEY 360 EF485035QKPSIHFIWGAHNPGNSNNATFKLILLSKSLQSIKGISTYTEAFKSLGKMMDIGDKAIEY  360************************************************************ NC_004108EEFCMSLKSKARSSWKQIMNKKLEPKQINNALVLWEQQFMVNNDLIDKSEKLKLFKNFCG  420EF485035 EEFCMSLKSKARSSWKQIMNKKLEPKQINNALVLWEQQFMVNNDLIDKSEKLKLFKNFCG 420 ************************************************************NC_004108 IGKHKQFKNNMLEDLEVSKPKILDFDDANMYLASLTMMEQSKKILSKSNGLKPDNFILNE 480 EF485035IGKHKQFKNNMLEDLEVSKPKILDFDDANMYLASLTMMEQSKKILSKSNGLKPDNFILNE  480************************************************************ NC_004108FGSKIKDANKETYDNMHKIFETRYWQCISDFSTLMKNILSVSQYNRHNTFRIAMCANNNV  540EF485035 FGSKIKDANKETYDNMHKIFETRYWQCISDFSTLMKNILSVSQYNRHNTFRIAMCANNNV 540 ************************************************************NC_004108 FAIVFPSADIKTKKATVVYSIIVLHKEEENIFNPGCLHGTFKCMNGYISISRAIRLDKER 600 EF485035FAIVFPSADIKTKKATVVYSIIVLHKEEENIFNPGCLHGTFKCMNGYISISRAIRLDKER  600************************************************************ NC_004108CQRIVSSPGLFLTTCLLFKHDNPTLVMSDIMNFSIYTSLSITKSVLSLTEPARYMIMNSL  660EF485035 CQRIVSSPGLFLTTCLLFKHDNPTLVMSDIMNFSIYTSLSITKSVLSLTEPARYMIMNSL 660 ************************************************************NC_004108 AISSNVKDYIAEKFSPYTKTLFSVYMTRLIKNACFDAYDQRQRVQLRDIYLSDYDITQKG 720 EF485035AISSNVKDYIAEKFSPYTKTLFSVYMTRLIKNACFDAYDQRQRVQLRDIYLSDYDITQKG  720************************************************************ NC_004108IKDNRELTSIWFPGSVTLKEYLTQIYLPFYFNAKGLHEKHHVMVDLAKTILEIECEQREN  780EF485035 IKDNRELTSIWFPGSVTLKEYLTQIYLPFYFNAKGLHEKHHVMVDLAKTILEIECEQREN 780 ************************************************************NC_004108 IKEIWSTNCTKQTVNLKILIHSLCKNLLADTSRHNHLRNRIENRNNFRRSITTISTFTSS 840 EF485035IKEIWSTNCTKQTVNLKILIHSLCKNLLADTSRHNHLRNRIENRNNFRRSITTISTFTSS  840************************************************************ NC_004108KSCLKIGDFRKEKELQSVKQKKILEVQSRKMRLANPMFVTDEQVCLEVGHCNYEMLRNAM  900EF485035 KSCLKIGDFRKEKELQSVKQKKILEVQSRKMRLANPMFVTDEQVCLEVGHCNYEMLRNAM 900 ************************************************************NC_004108 PNYTDYISTKVFDRLYELLDKGVLTDKPVIEQIMDMMVDHKKFYFTFFNKGQKTSKDREI 960 EF485035PNYTDYISTKVFDRLYELLDKGVLTDKPVIEQIMDMMVDHKKFYFTFFNKGQKTSKDREI  960************************************************************ NC_004108FVGEYEAKMCMYAVERIAKERCKLNPDEMISEPGDGKLKVLEQKSEQEIRFLVETTRQKN 1020EF485035 FVGEYEAKMCMYAVERIAKERCKLNPDEMISEPGDGKLKVLEQKSEQEIRFLVETTRQKN1020 ************************************************************NC_004108 REIDEAIEALAAEGYESNLEKIEKLSLGKAKGLKMEINADMSKWSAQDVFYKYFWLIALD1080 EF485035REIDEAIEALAAEGYESNLEKIEKLSLGKAKGLKMEINADMSKWSAQDVFYKYFWLIALD 1080************************************************************ NC_004108PILYPQEKERILYFMCNYMDKELILPDELLFNLLDQKVAYQNDIIATMTNQLNSNTVLIK 1140EF485035 PILYPQEKERILYFMCNYMDKELILPDELLFNLLDQKVAYQNDIIATMTNQLNSNTVLIK1140 ************************************************************NC_004108 RNWLQGNFNYTSSYVHSCAMSVYKEILKEAITLLDGSILVNSLVHSDDNQTSITIVQDKM1200 EF485035RNWLQGNFNYTSSYVHSCAMSVYKEILKEAITLLDGSILVNSLVHSDDNQTSITIVQDKM 1200************************************************************ NC_004108ENDKIIDFAMKEFERACLTFGCQANMKKTYVTNCIKEFVSLFNLYGEPFSIYGRFLLTSV 1260EF485035 ENDKIIDFAMKEFERACLTFGCQANMKKTYVTNCIKEFVSLFNLYGEPFSIYGRFLLTSV1260 ************************************************************NC_004108 GDCAYIGPYEDLASRISSAQTAIKHGCPPSLAWVSIAISHWMTSLTYNMLPGQSNDPIDY1320 EF485035GDCAYIGPYEDLASRISSAQTAIKHGCPPSLAWVSIAISHWMTSLTYNMLPGQSNDPIDY 1320************************************************************ NC_004108FPAENRKDIPIELNGVLDAPLSMISTVGLESGNLYFLIKLLSKYTPVMQKRESVVNQIAE 1380EF485035 FPAENRKDIPIELNGVLDAPLSMISTVGLESGNLYFLIKLLSKYTPVMQKRESVVNQIAE1380 ************************************************************NC_004108 VKNWKVEDLTDNEIFRLKILRYLVLDASMDPSDIMGETSDMRGRSILTPRKFTTAGSLRK1440 EF485035VKNWKVEDLTDNEIFRLKILRYLVLDASMDPSDIMGETSDMRGRSILTPRKFTTAGSLRK 1440************************************************************ NC_004108LYSFSKYQDRLSSPGGMVELFTYLLEKPELLVTKGEDMKDYMESVIFRYNSKRFKESLSI 1500EF485035 LYSFSKYQDRLSSPGGMVELFTYLLEKPELLVTKGEDMKDYMESVIFRYNSKRFKESLSI1500 ************************************************************NC_004108 QNPAQLFIEQILFSHKPIIDFSGIRDKYINLHDSRALEKEPDILGKVTFTEAYRLLMRDL1560 EF485035QNPAQLFIEQILFSHKPIIDFSGIRDKYINLHDSRALEKEPDILGKVTFTEAYRLLMRDL 1560************************************************************ NC_004108SSLELTNDDIQVIYSYIILNDPMMITIANTHILSIYGSPQRRMGMSCSTMPEFRNLKLIH 1620EF485035 SSLELTNDDIQVIYSYIILNDPMMITIANTHILSIYGSPQRRMGMSCSTMPEFRNLKLIH1620 ************************************************************NC_004108 HSPALVLRAYSKNNPDIQGADPTEMARDLVHLKEFVENTNLEEKMKVRIAINEAEKGQRD1680 EF485035HSPALVLRAYSKNNPDIQGADPTEMARDLVHLKEFVENTNLEEKMKVRIAINEAEKGQRD 1680************************************************************ NC_004108IVFELKEMTRFYQVCYEYVKSTEHKIKVFILPTKSYTTTDFCSLMQGNLIKDKEWYTVHY 1740EF485035 IVFELKEMTRFYQVCYEYVKSTEHKIKVFILPTKSYTTTDFCSLMQGNLIKDKEWYTVHY1740 ************************************************************NC_004108 LKQILSGGHKAIMQHNATSEQNIAFECFKLITHFADSFIDSLSRSAFLQLIIDEFSYKDV1800 EF485035LKQILSGGHKAIMQHNATSEQNIAFECFKLITHFADSFIDSLSRSAFLQLIIDEFSYKDV 1800************************************************************ NC_004108KVSKLYDIIKNGYNRTDFIPLLFRTGDLRQADLDKYDAMKSHERVTWNDWQTSRHLDMGS 1860EF485035 KVSKLYDIIKNGYNRTDFIPLLFRTGDLRQADLDKYDAMKSHERVTWNDWQTSRHLDMGS1860 ************************************************************NC_004108 INLTITGYNRSITIIGEDNKLTYAELCLTRKTPENITISGRKLLGARHGLKFENMSKIQT1920 EF485035INLTITGYNRSITIIGEDNKLTYAELCLTRKTPENITISGRKLLGARHGLKFENMSKIQT 1920************************************************************ NC_004108YPGNYYITYRKKDRHQFVYQIHSHESITRRNEEHMAIRTRIYNEITPVCVVNVAEVDGDQ 1980EF485035 YPGNYYITYRKKDRHQFVYQIHSHESITRRNEEHMAIRTRIYNEITPVCVVNVAEVDGDQ1980 ************************************************************NC_004108 RILIRSLDYLNNDIFSLSRIKVGLDEFATIKKAHFSKMVSFEGPPIKTGLLDLTELMKSQ2040 EF485035RILIRSLDYLNNDIFSLSRIKVGLDEFATIKKAHFSKMVSFEGPPIKTGLLDLTELMKSQ 2040************************************************************ NC_004108DLLNLNYDNIRNSNLISFSKLICCEGSDNINDGLEFLSDDPMNFTEGEAIHSTPIFNIYY 2100EF485035 DLLNLNYDNIRNSNLISFSKLICCEGSDNINDGLEFLSDDPMNFTEGEAIHSTPIFNIYY2100 ************************************************************NC_004108 SKRGERHMTYRNAIKLLIERETKIFEEAFTFSENGFISPENLGCLEAVVSLIKLLKTNEW2160 EF485035SKRGERHMTYRNAIKLLIERETKIFEEAFTFSENGFISPENLGCLEAVVSLIKLLKTNEW 2160************************************************************ NC_004108STVIDKCIHICLIKNGMDHMYHSFDVPKCFMGNPITRDMNWMMFREFINSLPGTDIPPWN 2220EF485035 STVIDKCIHICLIKNGMDHMYHSFDVPKCFMGNPITRDMNWMMFREFINSLPGTDIPPWN2220 ************************************************************NC_004108 VMTENPKKKCIALINSKLETQRDFSEFTKLMKKEGGRSNIEFD (SEQ. ID. No. 7)2263 EF485035VMTENPKKKCIALINSKLETQRDFSEFTKLMKKEGGRSNIEFD (SEQ. ID. No. 8) 2263*******************************************

APPENDIX 5 Medium genomic segment-amino acid sequence alignment ofLACV/human/1978 (Hughes et al. 2002) and LACV/human/1978 (this study)NC_004109 MICILVLITVAAASPVYQRCFQDGAIVKQNPSKEAVTEVCLKDDVSMIKTEARYVRNATG  60 EF485034MICILVLITVAAASPVYQRCFQDGAIVKQNPSKEAVTEVCLKDDVSMIKTEARYVRNATG   60************************************************************ NC_004109VFSNNVAIRKWLVSDWHDCRPKKIVGGHINVIEVGDDLSLHTESYVCSADCITGVDKETA  120EF485034 VFSNNVAIRKWLVSDWHDCRPKKIVGGHINVIEVGDDLSLHTESYVCSADCITGVDKETA 120 ************************************************************NC_004109 QVRLQTDTTNHFEIAGTTVKSCWFKSTTYITLDQTCEHLKVSCGPKSVQFHACFNQHMSC 180 EF485034QVRLQTDTTNHFEIAGTTVKSCWFKSTTYITLDQTCEHLKVSCGPKSVQFHACFNQHMSC  180************************************************************ NC_004109VRFLHRTILPGSIANSICQNIEIIILVTLTLLIFILLSILSKTYICYLLMPIFIPIAYIY  240EF485034 VRFLHRTILPGSIANSICQNIEIIILVTLTLLIFILLSILSKTYICYLLMPIFIPIAYIY 240 ************************************************************NC_004109 GIIYNKSCKKCKLCGLVYHPFTECGTHCVCGARYDTSDRMKLHRASGLCPGYKSLRAARV 300 EF485034GIIYNKSCKKCKLCGLVYHPFTECGTHCVCGARYDTSDRMKLHRASGLCPGYKSLRAARV  300************************************************************ NC_004109MCKSKGPASILSIITAVLVLTFVTPINSMVLGESKETFELEDLPDDMLEMASRINSYYLT  360EF485034 MCKSKGPASILSIITAVLVLTFVTPINSMVLGESKETFELEDLPDDMLEMASRINSYYLT 360 ************************************************************NC_004109 CILNYAVSWGLVIIGLLIGLLFKKYQHRFLNVYAMYCEECDMYHDKSGLKRHGDFTNKCR 420 EF485034CILNYAVSWGLVIIGLLIGLLFKKYQHRFLNVYAMYCEECDMYHDKSGLKRHGDFTNKCR  420************************************************************ NC_004109QCTCGQYEDAAGLMAHRKTYNCLVQYKAKWMMNFLIIYIFLILIKDSAIVVQAAGTDFTT  480EF485034 QCTCGQYEDAAGLMAHRKTYNCLVQYKAKWMMNFLIIYIFLILIKDSAIVVQAAGTDFTT 480 ************************************************************NC_004109 CLETESINWNCTGPFLNLGNCQKQQKKEPYTNIATQLKGLKAISVLDVPIITGIPDDIAG 540 EF485034CLETESINWNCTGPFLNLGNCQKQQKKEPYTNIATQLKGLKAISVLDVPIITGIPDDIAG  540************************************************************ NC_004109ALRYIEEKEDFHVQLTIEYAMLSKYCDYYTQFSDNSGYSQTTWRVYLRSHDFEACILYPN  600EF485034 ALRYIEEKEDFHVQLTIEYAMLSKYCDYYTQFSDNSGYSQTTWRVYLRSHDFEACILYPN 600 ************************************************************NC_004109 QHFCRCVKNGEKCSSSNWDFANEMKDYYSGKQTKFDKDLNLALTALHHAFRGTSSAYIAT 660 EF485034QHFCRCVKNGEKCSSSNWDFANEMKDYYSGKQTKFDKDLNLALTALHHAFRGTSSAYIAT  660************************************************************ NC_004109MLSKKSNDDLIAYTNKIKTKFPGNALLKAIIDYIAYMKSLPGMANFKYDEFWDELLYKPN  720EF485034 MLSKKSNDDLIAYTNKIKTKFPGNALLKAIIDYIAYMKSLPGMANFKYDEFWDELLYKPN 720 ************************************************************NC_004109 PAKASNLARGKESSYNFKLAISSKSIKTCKNVKDVACLSPRSGAIYASIIACGEPNGPSV 780 EF485034PAKASNLARGKESSYNFKLAISSKSIKTCKNVKDVACLSPRSGAIYASIIACGEPNGPSV  780************************************************************ NC_004109YRKPSGGVFQSSTDRSIYCLLDSHCLEEFEAIGQEELDAVKKSKCWEIEYPDVKLIQEGD  840EF485034 YRKPSGGVFQSSTDRSIYCLLDSHCLEEFEAIGQEELDAVKKSKCWEIEYPDVKLIQEGD 840 ************************************************************NC_004109 GTKSCRMKDSGNCNVATNRWPVIQCENDKFYYSELQKDYDKAQDIGHYCLSPGCTTVRYP 900 EF485034GTKSCRMKDSGNCNVATNRWPVIQCENDKFYYSELQKDYDKAQDIGHYCLSPGCTTVRYP  900************************************************************ NC_004109INPKHISNCNWQVSRSSIAKIDVHNIEDIEQYKKAITQKLQTSLSLFKYAKTKNLPHIKP  960EF485034 INPKHISNCNWQVSRSSIAKIDVHNIEDIEQYKKAITQKLQTSLSLFKYAKTKNLPHIKP 960 ************************************************************NC_004109 IYKYITIEGTETAEGIESAYIESEVPALAGTSIGFKINSKEGKHLLDVIAYVKSASYSSV1020 EF485034IYKYITIEGTETAEGIESAYIESEVPALAGTSIGFKINSKEGKHLLDVIAYVKSASYSSV 1020************************************************************ NC_004109YTKLYSTGPTSGINTKHDELCTGPCPANINHQVGWLTFARERTSSWGCEEFGCLAVSDGC 1080EF485034 YTKLYSTGPTSGINTKHDELCTGPCPANINHQVGWLTFARERTSSWGCEEFGCLAVSDGC1080 ************************************************************NC_004109 VFGSCQDIIKEELSVYRKETEEVTDVELCLTFSDKTYCTNLNPVTPIITDLFSVQFKTVE1140 EF485034VFGSCQDIIKEELSVYRKETEEVTDVELCLTFSDKTYCTNLNPVTPIITDLFSVQFKTVE 1140************************************************************ NC_004109TYSLPRIVAVQNHEIKIGQINDLGVYSKGCGNVQKVNGTIYGNGVPRFDYLCHLASRKEV 1200EF485034 TYSLPRIVAVQNHEIKIGQINDLGVYSKGCGNVQKVNGTIYGNGVPRFDYLCHLASRKEV1200 ************************************************************NC_004109 IVRKCFDNDYQACKFLQSPASYRLEEDSGTVTIIDYKKILGTIKMKAILGDVKYKTFADS1260 EF485034IVRKCFDNDYQACKFLQSPASYRLEEDSGTVTIIDYKKILGTIKMKAILGDVKYKTFADS 1260************************************************************ NC_004109VDITAEGSCTGCINCFENIHCELTLHTTIEASCPIKSSCTVFHDRILVTPNEHKYALKMV 1320EF485034 VDITAEGSCTGCINCFENIHCELTLHTTIEASCPIKSSCTVFHDRILVTPNEHKYALKMV1320 ************************************************************NC_004109 CTEKPGNTLTIKVCNTKVEASNAKVDAKPIIELAPVDQTAYIREKDERCKTWMCRVRDEG1380 EF485034CTEKPGNTLTIKVCNTKVEASNAKVDAKPIIELAPVDQTAYIREKDERCKTWMCRVRDEG 1380************************************************************ NC_004109LQVILEPFKNLFGSYIGIFYTFIISIVVLLVIIYVLLPICFKLRDTLRKHEDAYKREMKI 1440EF485034 LQVILEPFKNLFGSYIGIFYTFIISIVVLLVIIYVLLPICFKLRDTLRKHEDAYKREMKI1440 ************************************************************NC_004109 R (SEQ. ID. No. 9) 1441 EF485034 R (SEQ. ID. No. 10) 1441 *

APPENDIX 6Small genomic segment-Nucleoprotein (N) amino acid sequence alignment ofLACV/human/1978 (Hughes et al. 2002) and LACV/human/1978 (this study)NC_004110 MSDLVFYDVASTGANGFDPDAGYMDFCVKNAELLNLAAVRIFFLNAAKAKAALSRKPERK  60 EF485033MSDLVFYDVASTGANGFDPDAGYMDFCVKNAELLNLAAVRIFFLNAAKAKAALSRKPERK   60************************************************************ NC_004110ANPKFGEWQVEVINNHFPGNRNNPIGNNDLTIHRLSGYLARWVLDQYNENDDESQHELIR  120EF485033 ANPKFGEWQVEVINNHFPGNRNNPIGNNDLTIHRLSGYLARWVLDQYNENDDESQHELIR 120 ************************************************************NC_004110 TTIINPIAESNGVGWDSGPEIYLSFFPGTEMFLETFKFYPLTIGIHRVKQGMMDPQYLKK 180 EF485033TTIINPIAESNGVGWDSGPEIYLSFFPGTEMFLETFKFYPLTIGIHRVKQGMMDPQYLKK  180************************************************************ NC_004110ALRQRYGTLTADKWMSQKVAAIAKSLKDVEQLKWGKGGLSDTAKTFLQKFGIRLP (SEQ. ID. No. 11) 235 EF485033ALRQRYGTLTADKWMSQKVAAIAKSLKDVEQLKWGKGGLSDTAKTFLQKFGIRLP (SEQ. ID. No. 12) 235 *******************************************************

APPENDIX 7Small genomic segment-Nonstructural protein (NSs) amino acid sequencealignment of LACV/human/1978 (Hughes et al.) and LACV/human/1978 (this study)NC_004110 MMSHQQVQMDLILMQGIWTSVLKMQNYSTLLQLGSSSSMPQRPRLLSRVSQRGRLTLNLE  60 EF485033MMSHQQVQMDLILMQGIWTSVLKMQNYSTLLQLGSSSSMPQRPRLLSRVSQRGRLTLNLE   60************************************************************ NC_004110SGRWRLSIIIFLETGTTQLVTTILPSTDYLGI (SEQ. ID. No. 13)   92 EF485033SGRWRLSIIIFLETGTTQLVTTILPSTDYLGI (SEQ. ID. No. 14)   92********************************

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

We claim:
 1. A method of inducing an immune response against a JamestownCanyon virus infection in a mammal, comprising administering to themammal a therapeutically effective dose of an immunogenic compositioncomprising an infectious chimeric virus comprising a La Crosse virusgenome, wherein the G_(C) or G_(N) gene of the La Crosse virus genome issubstituted with the counterpart G_(C) or G_(N) gene from a JamestownCanyon virus.
 2. The method of claim 1, wherein the La Crosse virusgenome is derived from an LACV/human/1960 virus, an LACV/mosquito/1978virus, or an LACV/human/1978 virus.
 3. The method of claim 1, whereinthe G_(C) gene of the La Crosse virus genome is substituted with thecounterpart G_(C) and G_(N) gene of the Jamestown Canyon virus.
 4. Themethod of claim 1, wherein the infectious chimeric virus is inactivated.5. A method of inducing an immune response against a La Crosse virusinfection in a mammal, comprising administering to the mammal atherapeutically effective dose of an immunogenic composition comprisingan infectious chimeric virus comprising a Bunyaviridae genome, whereinone or both of the G_(C) and G_(N) genes of the Bunyaviridae genome issubstituted with one or both of the counterpart G_(C) and G_(N) genesfrom a La Crosse virus genome, wherein the La Crosse virus genomeencodes a G_(N) glycoprotein having a mutation at amino acid
 148. 6. Themethod of claim 5, wherein the mutated amino acid at position 148 ofG_(N) is alanine.
 7. The method of claim 5, wherein amino acid 148 ofG_(N) is deleted.
 8. The method of claim 5, wherein the mutated aminoacid at position 148 of G_(N) is any amino acid other than threonine. 9.The method of claim 5, wherein the La Crosse virus genome is derivedfrom an LACV/human/1960 virus, an LACV/mosquito/1978 virus, or anLACV/human/1978 virus.
 10. The method of claim 5, wherein the infectiouschimeric virus is inactivated.